Libro de Protocolos

PROTOCOLS
CELL COMMUNICATIONS LABORATORY
Edited by Virginia Monardes. July, 2003
PROTOCOLS INDEX
A: ANTIBODIES..............................................................................................................3
B: APOPTOSIS...............................................................................................................21
C: BIOCHEMISTRY.......................................................................................................32
D: LIPIDS BIOCHEMISTRY.......................................................................................106
E: CELL BIOLOGY......................................................................................................143
F: CELL CULTURE......................................................................................................181
G: PROTEIN FUSION..................................................................................................206
H: MOLECULAR BIOLOGY......................................................................................217
A: ANTIBODIES
WESTERN BLOTS...........................................................................................................4
PURIFICATION OF IG FROM SERA USING SEPHAROSE-PROTEIN A..................6
ELISA................................................................................................................................8
ELISA PROTOCOL..........................................................................................................9
ELISA PROTOCOL AQ..................................................................................................10
FACS PREPARATIONS..................................................................................................11
IMMUNOFLUORESCENCE.........................................................................................12
COUPLING PEPTIDES TO CARRIER PROTEINS.....................................................14
MOUSE MONOCLONALE ANTIBODY AFTER PEPTIDE INJECTION..................15
IMMUNOPRECIPITATION WITH T-CELL EXTRACTS............................................16
OVERLAY ASSAY.........................................................................................................17
OVERLAY ASSAY WITH PKC.....................................................................................18
ANTIBODY PURIFICATION FROM HYBRIDOMA SUPERNATANTS...................20
WESTERN BLOTS
November 1991
ALWAYS WEAR GLOVES
Make transfer buffer and place in freezer to chill.
Nitrocellulose preparation:
Remove roll and gently expose membrane -- it is very brittle when dry.
Cut a piece the size needed using a curved-bladed scalpel or a razor (For a minigel 2.5
in x 3.5 in)
Handle with flat surfaced forceps
Lay the cut nitrocellulose on buffer in a pan, first lay it on the surface and then allow
it it slip under the buffer. You may stack several pieces in the buffer.
Pad and sponge preparation:
Soak the pads (3M paper or Biorad transfer pads) and sponges in transfer buffer.
This buffer may be saved and reused many times for this purpose. It is
critical that there be no bubbles trapped in the pads and sponges.
Set up a stirrer and cover with paper to soak up any spills.
Place the tank with the transfer chamber on the stirrer, add a stirbar and 800 ml ice cold
transfer buffer. Make sure the stirbar spins freely. Add white plastic ice insert to the
back of the chamber. Orient the tank so that the ice is in back, and the red pole of the
transfer chamber is to your right. This means the black side of the transfer chamber is
to the back.
Whiteice
Clear Black
To prepare the sandwich:

1.) Transfer the soaked pads to the buffer tray with the open holder. Squeeze out all
bubbles. With the holder open so that the black side is down under the buffer
and the clear side is nearest you, place a sponge topped with a 3M pad on the
black back.
2.) Prepare the gel by a) removing the stacker - get rid of all of it because it will stick
to the nitrocellulose and make a huge mess. b) mark the lower right hand corner
of the gel for orientation, then c) using a spacer lift the gel and place it in the
same orientation on the lower end of the pad (this is so that when the sandwich
is turned upright and buffer drains out no air bubbles will be introduced around
the gel and prevent transfer.
3.) Using membrane forceps place the nitrocellulose directly over the gel, if
measured correctly it should fit well with space on all sides. No bubbles!
4
4.) Place another 3M pad on top of the nitrocellulose NO bubbles! followed by

another sponge. Align all the pads so they do not stick out the edges of the
holder (if they do the sandwich will not fit into the transfer apparatus)
Clear
Buffer
Sponge
3M pad
Nitrocellulose
Gel
3M pad
Sponge
Black
Place the first sandwich in the transfer holder with the black side of the holder next to the
black panel of the apparatus and prepare the second sandwich if it is to be used.
2 1
Sandwiches
White ice
Run at 30 V overnight for medium to largish proteins, less time for peptides, more for giant
proteins. It may also be run at up to 200 V for 1-2 hours.
.
To remove the blot from the holder:

1.) Turn off the current
2.) Open up the sandwich so the nitrocellulose is on top of the gel (black side down,
open the clamp towards you)
3.) Mark the corners of the gel on the nitrocellulose so you can see where the gel
was. This will be on the back of the blot. The prestained size markers will
prove that the transfer occurred.
4.) Transfer the nitrocellulose to a bath with blocker (usually 3% non-fat milk powder
in PBS with 0.02% NaN3.) Block for at least 2-4 hrs, overnight is better. Make
sure there is plenty of blocker so that the membrane is floating freely and the
shaker is on highish speed. (blocking solution may be reused.)
5.) Rinse briefly with PBS
6.) Incubate with first antibody 10 ml at whatever dilution is appropriate x 2 hr
7.) Wash 3 x 5-10 min with PBS
8.) Incubate with second antibody (Biorad horseradish peroxidase, 1:3000) x 2 hr
9.) Wash 10min with PBS
10.)Wash 10 min with 100 ml PBS with 2 drops Tween 20
11.)Wash 10 min with PBS
12.)Add developer made fresh just before using. If the blot has not developed within
10 min you may add another 10 l H202. Rinse extensively with water.
Membranes may be stored dry wrapped in a Kimwipe and foil.
5
PURIFICATION OF IG FROM SERA USING SEPHAROSE-PROTEIN A

Material
Saturated (NH4)2SO4 : at 25oC saturation for 761 g/l (4.1 M)
Tris Buffer pH 8.5
0.1 M Tris
12.12 g
0.150 M NaCl
8.77 g
2.5 mM NaN3
0.16 g
H2O
to 1 liter
(for frequent experiments make a 10x concentred Buffer)
Dialysis tubing : Spectrapor 4, M.W. Cutoff 12-14.000

10x PBS pH 7.0-7.5
Na2HPO4 2H2O
KHPO4
NaCl
H2O
89 g
13.6 g
58 g
to 1 liter
Glycine 0.1 M-HCl Buffer pH 3

Protein A Sepharose CL-4B (Pharmacia No. 17-0780-01)
Tris-HCl pH 8.8
1.5 M Tris
Tris-HCl pH 6.8
0.5 M Tris
30% acrylamide/bisacrylamide (38 : 2)

20% SDS
TEMED
10% APS
a) (NH4)2SO4 precipitation of Ig from sera
While the serum is being stirred gently on ice with a magnetic bar, add sarurated
ammonium sulfate dropwise to bring a final concentration of
50 %. Alow sufficient time
for precipitation by stirring for 6 hr or overnight at 4 C.
Centrifuge at 8000 RPM (3000x g) in the Kontron (rotor A 8.24) for
30 min.
Carefully remove and save the supernatent for gel analysis. The pellet is resuspended in a
minimum volume of Tris Buffer pH 8.5 (50 ml Tris for every 20 ml of starting serum).
Mesure OD 280 before dialysis.
b) Dialysis of resuspended proteins after ammonium sulfate fractionation
Dialyse 3x times in 10 volumes of Tris Buffer pH 8.5 for 2 hr, last time overnight at 4oC.
Mesure OD 280 after dialysis.
c) Affinity chromatography on sepharose-protein A column
6
Swell the beads in 1x PBS and pull them in an appropriate column.

1.5 g of dry beads gives 5 ml of swollen gel and can fixe 100 mg of pure Ig.
Wash the column with 20 volumes of Tris Buffer pH 8.5.
When all the Buffer has passed, gently charged the dialysed suspended proteins on the
column. Collect 3ml fractions with a slow debit until all the suspention has penetrated the
gel. Then eluate the non interacting proteins with Tris Buffer pH 8.5 with a fast debit,
collecting 3ml fractions and measuring OD 280.
When OD 280 is down to baseline, eluate the fixed Ig with the Glycine buffer pH 3. Collect
3ml fractions into tubes already containing 1.5 ml of Tris Buffer pH 8.5 to immediately
neutralise the acidic pH of the elution buffer. Monitor OD 280 to check when elution is
complete.
Regenerate the column by passing 10 volumes of Tris Buffer pH 8.5, 10 volumes of NaAcNaCl Buffer pH 4.5 and then again 10 volume of Tris Buffer pH 8.5 through the column.
Store the column at 4oC in Tris Buffer pH 8.5 in the presence of 0.3 % NaN3.
d) Mini SDS-PAGE analysis of the Ig purification
Check purification by SDS-PAGE analysison a 10 % mini gel.
Load 1-5 g per lane of the proteins before purification, the non-interacting proteins in flow
through and wash fractions and the purified proteins.
Run the purified Ig fraction under both reducing and non-reducing conditions and compare
with a standard IgG fraction containing both the heavy and light chain (e.g Bio Rad G_R
IgG (H+L) ).
Also add a molecular weight marker.
ELISA
1) Let dry 10 l of antigen per well, placing the plates in dessicator overnight.
For pure Ag use approx. 10 ng/well.
2) Rinse the plate with PBS and saturate free sites with PBS-1% BSA-0.05% Tween 20.
Incubate 1 hr at 37oC.
3) Rinse again with PBS and keep the plate moiturized until use. Store at 4oC.
4) First row of the plate is used as a blank so, it has to be incubated with 50 l/well of the
solution in where the antibodies are (control medium). Add 50 l of the first antibody
(hybridoma supernatant) and incubate for 1 hr at 37oC.
5) Wash the plate with PBS-0.05% Tween 20. 5 x 5 min.
6) Add 50 l of the second antibody diluted in blocking solution (a-mouse IgG-HRP diluted
1:500). Incubate 1 hr at 37oC.
7) Wash as indicated in #5.
8) Incubate with 100 l of substrate solution (15-60 min, room temperature). Prepare
solution immediately before use.
0.504g Citric acid
1.367g Na2HPO4.7H2O
100 ml MQ-water, pH 5.0-5.2
Per 10 ml of this buffer add 0.02g o-phenylenediamine.2HCl (OPD) (light sensitive
and mutagenic),
and immediately before use add 6.7 l of 30% H2O2.
9) Read in ELISA-reader at 450 nm.
ELISA PROTOCOL
(after the protocol used in N. Fasel's Lab, 1995)
1. Coat each well to be used on plate with 100 l antigen solution (take 100 ng to 10 g
protein or peptide per well; 0.5 g of peptide seems to work well).
2. Wrap box and leave overnight at 4 C.
3. Block unspecific binding by addition of 100 l 2x blocking solution per well, incubate
for two hours at room temperature.
Meanwhile the dilution of the serum/lysate to be tested in antibody dilution solution can
be prepared and left at room temp. for 30 min.
4. Remove blocking solution by virtually "throwing" liquid content away and remove
remaining drops by banging uspide-down against the bench sufrace covered with paper
towel. Wash four times with 100 l of 1x PBS.
5. Put 100 l of the preincubated antibody/lysate-mix into each well and incubate for 1 hr
at RT. (Dilution series: Put 100 l of antibody dilution buffer into each well; add to well
supposed to contain the highest concentration 100 l of the 1:10 diluted serum, mix
well. Take 100 l from this well and add to the next well, mix, take 100 l and so on).
6. Discard the solution and wash wells four times with 1x PBS, discard each time.
7. Put 100 l of the diluted 2nd antibody solution into each well and leave 1hr at RT.
8. Wash four times with 100 l PBS, discard each time.
9. Put 100 l of NPP-containing developer solution into each well and leave at RT until
yellow colours appears (10 min.-60 min).
10. Measure absorption at 405 nm.
Solutions/Material:
1x PBS
used to dilute antigen coating solution
2x blocking solution 1xPBS, 5% milk powder, 0.05% Tween20

antibody dilution
1xPBS, 2.5% milk powder, 0.05% Tween20 solution
2nd antibody
Polyclonal anti mouse (anti rabbit etc.) IgG coupled to alkaline

phosphatase (SIGMA, dil 1:1000)
Developer solution
1M Diethanolamin (pH=9.8), 1 mM MgCl2 (before

development Nitrophenyl-phosphate pills are added
to a final conc. of 1 mg/ml)
Plates: NUNC ImmunoPlates, MaxiSorp F96
ELISA PROTOCOL AQ
Protocol for ELISA detection of proteins and peptides.
Incubate plates with 100ul of 10ug/ml antigen solution (either ovalbumin or
hemocyanin) for about 1 hour. With peptides use about 100-200 ng per well. Incubate
overnight at 37C. Leave wells of the first column balnk.
Wash plates with PBS, then block for 1 hour at 37C with PBS containing 1% BSA
and 0.05% Tween 20. Use 200ul per well.
Rinse again with PBS and keep plate moisturized until use. Store at 4C.
The first column of the plate is always used as a blank, so nothing is coated there
and it is incubated with 100ul per well of the solution in which antibodies are diluted. Each
row is incubated with the appropriate antibodies diluted as desired. To test anti-sera, serial
dilutions in steps of 2 starting with a dilution 1:10 generally works well. Incubate 1 hour at
37C.
Wash the plate with PBS containing 0.05% Tween 20, 5 x 5 min.
Add 100 ul second antibody diluted in blocking buffer. Incubate 1 hour at 37C.
Wash as above.
Incubate with 100ul of substrate solution. Prepare solution immediately before use:
0.504g citric acid
1.367g Na2HPO4.7H2O
100 ml Ultrapure water, pH 5.0-5.5
Per 10ml of this buffer add 0.02g o-phenylenediamine.2HCl (OPD) (light sensitive,
mutagenic) and immediately before use add 6.7ul of 30% H2O2.
Read in Bio-Rad ELISA reader at 450 nm at various time intervals.
Comments:
Best second antibodies to rabbit (H+L chains) from Kirkgaard & Perry. Others from
Bio-Rad also work. Protocol can be modified to detect protein-protein interactions (see
Ghosh et al., 1994)
AQ, 2-8-97
10
FACS PREPARATIONS
A) Intracellular staining
Solutions
Sol A : 1xPBS/2.5% FCS/0.05% NaN3
Sol B 10x : 10x PBS/0.02% NaN3/1% saponin
Sol B 1x
1e antibody : diluted in Sol A
Take 9 V antibody + 1 V sol B 10x
Amount needed : 100 l for each reactions
Test different concentrations (typically a serum must be diluted 100-1000x)
2e antibody : diluted as fallowed
Suspend 2 mg antibody (FITC or DTAF conjugated, Jackson ImmunoResearch) in 1.5 ml
steril H2O. Leave for 1-2 hours at RT on a rotative shaker protected from light. Stock
aliquots at -20 C.
For one FACS experiment :
Antibody
Sol. B 10x
Sol. A
5_
100_
895_
Method
1. Count 105-106 cells for each experiment.
2. Wash them 2 times with PBS.
3. Resuspend the cells in 1 ml 4% paraformaldehyde + 10 l Sol. B 10x and fixe them for
10 minutes at RT.
4. Wash 2 times with PBS.
5. Add 100 l of first antibody for each experiment and incubate for 30 minutes at RT.
6. Wash 3 times with sol. B.
7. Add 100 l of second antibody for each experiment and incubate for 30 minutes at RT.
8. Wash 3 times with sol. B.
9. Suspend the cells in 100-300 l PBS for FACS.
10. If cells have to be keep more than 3 hours fixe them for 10 minutes in 1 ml 4%
paraformaldehyde. Then wash them 3 times with PBS and suspend them in 100-300 l
of sol. A for FACS.
11
IMMUNOFLUORESCENCE
Preparation of Swiss 3T3 fibroblasts: adherent cells. Cells grown on coverslips.
Coverslips (Microscope Cover glasses, 14 mm;
Coverslips are stored in 70% ethanol at 4C. They are flamed and put in a 10 cm Nunc dish.
Cells are allowed overgrow the cover slips and stimulated as described in the "cell culture"
protocol.
Remove the medium (with or without stimulator).
Fixation: Incubate with paraformaldehyde 4% for 30 minutes at RT. Be careful,
paraformaldehyde is highly toxic.
Wash 5 x 3 minutes with PBS at RT.
Permeabilization: 10 minutes with 0.2% Triton X-100 in PBS at RT.
Wash 5 x3 minutes with PBS at RT.
Block for 30 minutes at 37C with 4% BSA (Sigma; A-2153) in PBS.
First antibody:
- Overnight at 4C
- 3 hours at RT
or:
Put a piece of parafilm in a box containing humid paper and put 10 l of antibody solution
on parafilm. Put the coverslips on drops.
Dilution for all antibodies:
1:50 in blocking solution.
- mouse IgM anti-caveolin-1 (monoclonal; Transduction Laboratories,
C13620)
- rabbit anti-PKC (polyclonal; Sigma: P4334 (PKC), P8333 (PKC), P8458

(PKC) and P0713 (PKC).
Wash 5 x 3 minutes with PBS / 0.01% Tween (Sigma, P-1379) at RT.
Secondary antibody:
1 hour at 37C protected from light.
Put 10 l of antibody solution on parafilm in a humidified box.

Dilution: 1:400 in blocking solution
- anti-mouse-Cy3 (Jackson, 115-165-144)
12
- anti rabbit- FITC (Jackson, 111-095-144)

Wash 5 x 3 minutes with PBS / 0.01% Tween at RT.
Rinse briefly in dH2O to remove salt.
Mount in FluorSaveTM Reagent ( Calbiochem, 345789) on glass slides which were
previously cleaned with ethanol 70%.
Store at 4C protected from light.
Analysis by confocal microscopy (Zeiss).
Wavelength for FITC:
for Cy3:
488 nm
543 nm
Preparation of 4% paraformaldehyde:
Warm PBS at 65C in water bath. Place everything in ventilated hood. 20 g of pformaldehyde (Fisher) are added to the warm PBS and let stir for over 1 hour. It does not
need pH adjustment, but check with pH paper that it is around 7. Add more PBS to
complete volume to 500 ml and aliquot in 50 ml tubes.
Store at -20C for several months. Solution may be thawed several times.
13
COUPLING PEPTIDES TO CARRIER PROTEINS

Protocol for activation of any protein (example: Limulus Polyphemus Hemocyanin)
to permit subsequent reaction with free -SH groups present in cysteines of peptide to be
coupled.
Make 1.5 mls of Limulus Polyphemus Hemocyanin (Sigma H1757) solution at
20mg/ml in 50mM NaPi buffer pH7
Put a 1 ml aliquot without creating foam into an Eppendorf tubes
Add 20ul MBS (Sigma M8759) dissolved at 25 mg/ml in dimethyl-formamide
(Fluka, puriss).
Incubate protein for 1 hour at room temperature. Then load 1ml of solution onto a
gel filtration column (Swift TM, Pierce) equilibrated in NaPi buffer, calibrated to permit
separation of activated protein from MBS. Collect 1ml fractions and pool protein peak,
about 4 ml volume (fractions 6-10). Re-run protein peak over column; collect again 1ml
fractions; pool the 4 protein peak fractions as activated KLH (enough for 2-3 coupling
reactions).
To an Eppendorf tube containing 7.5 mg of peptide add 1.5 mls of activated LPH
solution (final peptide conc. about 2.5 mM). Leave standing at room temperature for 30
min., then put on rotating shaker overnight. In most cases a precipitate will form
immediately. Sometimes the peptide will dissolve, then slowly turn opalescent and
precipitation will occur later on.
Next day, the product of each coupling reaction is mixed with 3ml of PBS and split
up into 5 portions of 0.9ml. One such portion is mixed 1:1 with Freunds complet or
incomplete adjuvant for subcutaneous injection into rabbits.
Comments:
Same protocol has been applied to couple peptides to ovalbumin. The most crucial
step is getting good separation of activated carrier protein and free MBS, otherwise the
latter will prevent peptide coupling later on.
AQ, 2-8-97
14
MOUSE MONOCLONALE ANTIBODY AFTER PEPTIDE INJECTION

Material
Young BALB-C mice
Peptide diluted 2 mg/ml : MAP, reverse-MAP or free peptide
Adjuvant Freund Incomplete (AFI)
1 and 2 ml syringes : Becton Dickinson
Orange needles : Becton Dickinson 25G5/80.5x16
Box for injection (GP's lab)
Methode
Mix peptid and AFI in a 1 : 1 ratio in parafilm sealed 2 ml syringe .
Sonicate the mix briefly 2-3 times until you get a nice emulsion.
Inject the content of the syringe into a 1 ml sirynge.
Eventually put the syringe vertically in the frige to allow air bubbles to go out.
Inject with an orange needle 50 l (50 g) of the emulsion (see below the scheme for
injection). The emulsion provokes a swelling at the base of the tail.
Tail cross section
veins
inervated regions
point of injection
15
IMMUNOPRECIPITATION WITH T-CELL EXTRACTS

The T-cell extracts (total volume 1ml) in Crumptons lysis buffer (CLB) containing
10 mM iodoacetamide and protease inhibitors (PMSF, 50uM final conc.) are first pretreated
with Pansorbin in round-bottomed Falcon 2063 tubes for 2 hours at 4C, and then after
removing the supernant (spin 10min., 2500 rpm) over night at 4C with a fresh aliquot of
washed Pansorbin.
The supernatant is treated next day at RT for 1 hour with 50ul Protein A beads
(Pharmacia # 17-0780-01). At this point the remaining supernatant is split into x batches
(depending on number of IP exp.) corresponding to roughly 107/x initial T-cells.
To each volume of 500ml pre-treated cell extract 500ml CLB and 10ul or more of
affinity purified IgGs are added and incubated over night at 4C. Depending on the
affinity of the antibody, this incubation may be reduced to 2-3 hours.
Next day add 50ul ProtA beads and incubate for 1 hour at RT. Then
remove
supernatant and wash beads 6x with wash buffer in small Eppendorf tube at RT. Spin each
time 1min. at 1000rpm in Eppendorf centrifuge.
The proteins specifically bound are eluted from the beads with sample buffer, 1min. at
100C, in the presence of 1M DTT and 2M iodoacetamide to alkylate thiol groups
The supernatant from the initial IP can be reused to immunoprecipitate another antigen,
as long as it did not coprecipitate with the first. To remove residual ProtA beads this
supernatant must be filtered though 0.2um filters (Schleicher & Schuell, FP030/3). Then
the process is repeated, starting with the incubation with the antibody.
COMMENTS/OBSERVATIONS
AQ 17-5-95
SOLUTIONS
Crumptons Lysis Buffer:
0.5% NP-40, 0.5% Deoxycholate, 50mM NaCl,

25mM Tris-HCl pH=8.2
Wash buffer:
0.5% NP-40, 500mM NaCl, 10mM

EDTA, 125mM Tris-HCl pH 8.2.
PanSorbin
(Calbiochem. #507858)
Place 2.5ml of 10% Pansorbin into a round-bottomed Falcon

2059 tube. Add 6ml CLB and mix. Spin at RT, 10min., 2500
rpm. Discard supernatant. Resuspend pellet in 6ml CLB, mix
and repeat centrifugation. Resuspend pellet again in 6ml
CLB, distribute 6x1ml aliquots into round bottomed F2063
Falcon tubes. Spin as above and store tubes at 4C until
needed.
16
OVERLAY ASSAY
Run SDS-PAGE using Bio-Rad mini-gel system, loading max. 50-100 ug of protein
per lane. For lung extracts, as little as 10-20 ug protein are required to get a signal in the
overlay assay, for other tissues or extracts from cultured cells as much protein as possible
should be loaded.
Transfer to nitrocellulose at 30V over night. Cool unit to 4 C on ice.
If necessary, cut blot into strips (stain region corresponding to gel top using Ponceau
Red S or Amid Schwarz). Block at least for 24 hours in block solution at 4 C.
Wash for 15-30 min. in PBS.
Then incubate with GST-PKC in fusion protein dilution buffer at 10ug/ml for 2
hours in cold room at 4 C on tilting shaker.
Wash 3x 10 min. at room temperature on rotating shaker (Tschopps lab) in large
volumes of PBS (AQs version)/0.04% NP-40.
Incubate with first antibody (1Ab), rabbit-anti-GST (serum#218) diluted 1:2000 in
1Ab dilution buffer, for 1.5 hours on rotating shaker (Tschopps lab).
Wash 3x 10 min. in PBS (AQs version) at room temperature on rotating shaker
(Tschopps lab).
Incubate with 2Ab, Goat-anti-rabbit IgG HRPO coupled (Bio-Rad) diluted 1:3000
in 2Ab dilution buffer, for 1.5 hours on rotating shaker (Tschopps lab) at room
temperature.
Wash 3x PBS (AQs version). Second wash should contain 0.04% NP-40.
Develop at room temperature with chloronaphtol/hydrogen peroxide in PBS, i.e. 9
ml PBS (AQs version), 1 ml chloronaphtol stock (stored at -20C), 10 ul H2O2 (30%
conc., stored at 4C, light-protected).
Comments:
Results/Conclusions:
Solutions for Overlay assay:
AQs 10x PBS - 8.5g NaCl(Mw 58.4)/8.8g NaHPO4 (Mw 142)/1.9g NaH2PO4.H2O (Mw
138) in 1l final volume. pH of 1x PBS should be around 7.2.
Block solution - 3.5% milk powder in PBS (AQs version) with 2mM sodium azide.
GST-PKC fusion protein dilution buffer - To PBS with 0.04% NP-40 add 10% ethylene
glycol.
1Ab dilution buffer - PBS with 10mg/ml BSA. Add 2mM sodium azide for antibody
storage.
2Ab dilution buffer - PBS with 10mg/ml BSA. Add NP-40 to 0.04% final concentration.
Do not add sodium azide.
Chloronaphtol stock - 30 mg of chloronaphtol (Peroxidase developer from Bio-Rad) per
10ml of ethanol.
AQ, 4-1-95
17
OVERLAY ASSAY WITH PKC

Run SDS-PAGE using Bio-Rad mini-gel system, loading max. 50-100 ug of protein
per lane. For lung extracts, as little as 10-20 ug protein are required to get a signal in the
overlay assay, for other tissues or extracts from cultured cells as much protein as possible
should be loaded. For purified proteins 1-2 ug total protein are sufficient.
Transfer to nitrocellulose at 30V over night. Cool unit to 4 C on ice.
If necessary, cut blot into strips (stain region corresponding to gel top using Ponceau
Red S or Amid Schwarz). Block at least for 24 hours in block solution at 4 C. Wash for 1530 min. in PBS.
Then incubate with PKC (Threonine Sepharose fraction 19-20, ......) in
phosphorylation buffer (+/- ATP, +/- PDBu) for 1.5-2 hours RT on tilting shaker lab F413.
Make PKC solution as indicated below
Phosphorylation buffer- Stock Solutions:
3 % Triton X-100: Dilute Pierce Surfact-Amp (28314 G) 10% to
3%
with
deionized water. This is 43 mM. For 5ml of a 10x stock use 1500ul; dilute to final vol. 5ml
with ddH2O.
Phosphatidyl serine: Stock is 20 mg/ml (31 mM), mol. wt. 820, dioleoyl
phosphatidyl
serine from Avanti Polar Lipids. For 5ml of a 10x stock at 10 mol%, dry down 696 ml and
resuspend in the 5ml of Triton X-100 3%
Dioctanoyl glycerol (diC8): Stock is 25 mg/ml (73 mM), mol. wt. 344, from Avanti Polar
Lipids. For a 2 mol% stock, dry down 60 ul of DiC8 and resuspend in 5 ml of above PS
/TX100 mix. This solution will actually be only 1.2 mol% sn-1,2-DiC8, the rest is inactive
sn-1,3-diC8 as determined by DGK assay.
Working lipid mix: Dry PS in a glass disposable tube to make a final concentration of 10
mol % in 5 ml 3% Triton X-100. Use PS stock to dissolve diC8 to a final concentration of 2
mol %. Mix well with a vortex and allow to sit in a 37 oC bath for a few minutes, mix well
again. Make 0.5ml aliquots and freeze at -20 oC. Dilute each aliquot to a final volume of 5
ml for phosphorylation on blots. Final assay concentration will be 0.3% TX-100, 10mol%
PS and 1.2 mol% sn-1,2-diC8.
PKC overlay experiment: To one such lipid aliquot (0.5ml), add the following reagents
0.5 ml 200 mM Tris, pH 7.5
0.5 ml 50 mM MgCl2
0.5 ml 2 mM CaCl2
1 ml Thr. Seph. PKC peak (prep. 1 brain)
2 ml ddH2O
After PKC incubation, wash 3x 10 min. at 4oC on rotating shaker (cold room) in
large volumes of PBS(AQs version)/0.04% NP-40.
Incubate with first antibody (1Ab), rabbit-anti-PKCg (Boehringer Mannheim)
diluted 1:500 in 1Ab (AQs version) dilution buffer, for 1.5 hours on tilting shaker (F413).
Wash 3x 10 min. in PBS (AQs version) at 4oC on rotating shaker (cold room),
second wash with 0.04% NP-40.
Incubate with 2Ab, Goat-anti-rabbit IgG HRPO coupled (Bio-Rad) diluted 1:3000
in 2Ab dilution buffer (AQs version), for 1.5 hours on tilting shaker (F413) at room
temperature.
Wash 3x PBS (AQs version) in cold room. Second wash should contain 0.04% NP40.
Develop at room temperature with chloronaphtol/hydrogen peroxide in PBS, i.e. 9
ml PBS (AQs version), 1 ml chloronaphtol stock (stored at -20C), 10 ul H2O2 (30%
18
conc., stored at 4C, light-protected).

NB. Also ECL can be used to develop overlay blots, but washes must be more
stringent, at least 4x7 min. at each step where washing is required
Comments:
Solutions for Overlay assay:
AQs 10x PBS - 8.5g NaCl(Mw 58.4)/8.8g NaHPO4 (Mw 142)/1.9g NaH2PO4.H2O (Mw
138) in 1l final volume. pH of 1x PBS should be around 7.2.
Block solution - 3.5% milk powder in PBS (AQs version) with 2mM sodium azide.
GST-PKC fusion protein dilution buffer - To PBS with 0.04% NP-40 add 10% ethylene
glycol.
1Ab dilution buffer - PBS with 10mg/ml BSA. Add 2mM sodium azide for antibody
storage.
2Ab dilution buffer - PBS with 10mg/ml BSA. Add NP-40 to 0.04% final concentration.
Do not add sodium azide.
Chloronaphtol stock - 30 mg of chloronaphtol (Peroxidase developer from Bio-Rad) per
10ml of ethanol.
AQ, 26-3-97
19
ANTIBODY PURIFICATION FROM HYBRIDOMA SUPERNATANTS

Washing buffer: PBS 1x
Elution buffer: 0.1M Glycine pH 3.0/ 0.5M NaCl (1.88g Glycine + 25ml 5M NaCl for 250
ml)
Buffers need to be cold before starting
1) Equilibrate the column (2ml of anti-rat Agarose, Sigma) with 50 ml of PBS
2) Do ammonium sulfate cut of 500ml of supernatant and dialyze with PBS o.n. at 4C
2) Load the column with the dialyzed antibody. Recover the flow through.
3) Wash the column with 100ml of cold PBS.
4) Prepare tubes for elution: Add 200l of 1M Tris pH 7.4 per tube and labelled them
5) Elute the column with Elution buffer and collect fractions of 2 ml.
6) Mix the eluate with the Tris after every tube collection
7) Monitor for protein at OD=280nm and pool those fraction containing the antibody
The column should be washed immediately with 80ml of PBS and 20ml of PBS containing
azide if is not needed the same day.
With the flow through repeat steps 2-7 until no more Ab binds to the column
8)Concentrate and dialyze the antibody using Centricon 30 (Amicon)
(Tube#) A 280nm
1-------------2-------------3-------------4-------------5-------------6--------------
7-------------8-------------9-------------10--------------
20
B: APOPTOSIS
ANALYSIS OF APOPTOTIC CASPASE-3 (FLUOROMETRIC)......................................22

ANALYSIS OF APOPTOTIC DNA FRAGMENTATION ON AGAROSE GEL...............23
DNA CONTENT ASSAYS BY FLOW CYTOMETRY.......................................................24
IN SITU CASPASE-3/CASPASE-8 ASSAY BY FACS.......................................................26
VIABILITY ASSAYS BY FACS..........................................................................................27
ASSESSMENT OF CHROMATIN CONDENSATION AND MORPHOLOGICAL
CHANGES............................................................................................................................28
QUANTIFICATION OF PHOSPHATIDYLSERINE EXPOSURE BY FACS...................29
DNA FRAGMENTATION ASSAY......................................................................................30
CASPASE-3 ACTIVITY ASSAY (CHROMOGENIC)........................................................31
21
ANALYSIS OF APOPTOTIC CASPASE-3 (FLUOROMETRIC)

References
- M.P. Boldin et al. (1996) Cell 85, 803-815.
- H.R. Stennicke and G.S. Salvesen (1997) JBC 272(41), 25719-25723.
Materials and solutions
For this assay, a fluorometer (eg Fluoroskan II) and special black ELISA plates (96 wells)
are needed.
Ac-DEVD-AMC, as a powder from Alexis, is dissolved in PBS to a final concentration of
10 mM (200x conc.) and stored in aliquots of 50-100 l at -20 C.
CHAPS buffer: 5 mM CHAPS detergent, 2 mM MgCl 2, 150 mM NaCl, 5 mM EDTA, 10
mM Tris-HCl (pH=7.4), 2 mM DTT.
The assay resides on the fact that caspase-3 recognizes specifically the tetrapeptide motif
DEVD. In the assay, this tetrapeptide is coupled to amino-methylcoumarine (AMC), which
uncleaved is only slightly fluorescent. Upon liberation by caspase-3 cleavage, AMC
becomes highly fluorescent, and allows thus quantification of caspase-3 activity. I did
measurements with the B lymphoma cells A20. Upon incubation with 100 ng/ml FasL,
first caspase-3 activity was observable after about an hour.
Protocol for A20 cells and Fluoroskan II:
Per condition, 2 mio cells, preferentially in duplicate, are taken (1-3 mio should work). The
cells are incubated either in 24 well plates at 37 C, 5% CO 2 (especially for incubations
longer than 3 h) or in 1.5 ml tubes in a 37 C waterbath (up to 3 h, with resuspension of
cells every hour). Do not forget to switch on/prewarm fluorescence about 1 h before
measurement!
Then the cells are harvested by centrifugation 3 min. at 3000 rpm at 4 C. The cells are
washed once with ice-cold PBS and are then lyzed in 100 l CHAPS buffer supplemented
with 200xDEVD-AMC solution to a final concentration of 50 M (100 M does also
work). Incubation for 30 min. at 37 C. Then the samples are spun for 10 sec. at 13'000 rpm
to get rid of the unlyzed material. The supernatant is pipeted into wells of a black ELISA
plate. Try to pipet to leftmost wells (starting with A1) to minimize scanning time.
The prewarmed machine is set to the preset excitation and emission wave-lengths "1"
(excitation 355 nm, emission 460 nm), the plate is put into position. Measurement is started
by "Start" button. If reading is not stopped, results are printed automatically, otherwise you
can print them by pushing "1". Results can be read between 30 min and 2 h. Be aware of
the upper detection limit of the machine, so it is preferable to take measurements at
different time points!
After usage rinse plate several times with normal and then with deionized water. Turn off
the Fluoroskan.
M. Hunn/ Aug. 98
22
ANALYSIS OF APOPTOTIC DNA FRAGMENTATION ON AGAROSE GEL

The dissolution of the nuclear membrane is a rather late event observed during apoptosis. In
this way, DNAse I, which normally resides in the endoplasmatic reticulum, gains access to
the nuclear DNA, cleaves the DNA between the nucleosomes, and creates thus fragments
with a length of 180 bp or multiples of 180 bp. When these fragments are analyzed on an
agarose gel (2%), a typical ladder-like pattern is observed in many cases. This is a typical
sign of apoptotic cell death.
This protocol is adapted to the B lymphoma cell line A20.
Two plates containing 1 mio cells in 4 ml medium are prepared. To those plates 2 l of 100
g/ml FasL (final 50 ng/ml) is given. Then the cells are incubated for 4 h at 37 C, 5% CO2.
The cell are then harvested: Spin cells in three 1.5 ml tubes (1 min. at 5000 rpm).
Resuspend the individual cell pellet in about 300 l of PBS and put in a single tube, spin
cells. Then the cell pellet is resuspended in 100 l PBS, and cells are lyzed by addition of
100 l phenol/chloroforme/isoamylalcohol (25:24:1). Vortex. Centrifuge the cells for 30 sec
at 13000 rpm. Transfer 50 l of the water phase to a new tube.
Of this crude DNA preparation, 17.5 l are mixed with 2 l buffer H (BM: for restriction
digests) and 0.5 l RNAse A (500 g/ml). Incubation 30 min. at 37 C. Analyze the DNA
on a 2% agarose gel.
M. Hunn/ Aug. 98 (acc. to P. Schneider, TP)
23
DNA CONTENT ASSAYS BY FLOW CYTOMETRY

Purpose of the test: Quantify hypodiploid cell populations using propidium iodide (PI).
Materials
-PI Stock solution 1mg/ml ( dangerous!- this compounds is carcinogenic and
mutagenic)
-Filterd PBS and FBS fetal bovine serum.
- Isotonic buffer
-5 ml FACS tubes (Falcon N Cat 352052).
-Methanol PA cold (-20C)
Methods:
1. Seed cells in a 24- or 6-well plate (options determined by experiment) in order to
obtain at least 2*105 cells for evaluation.
2. For adherent cells, put the medium of each well in 1.5 ml eppendorfs tubes. Then
add 100l of trypsin (for 24 wells plate) over the cells and leave at 37C until cells
detach.
Note: When cells have been starved, add 100ul of FBS to cell samples to neutralize
trypsin and avoid cell damage.
3. Centrifuge the cells for 7 min at 3000 r.p.m.
4. The pellet is resuspended vigorously by hand and tubes are kept cold on ice.
5. The cells are resuspended in methanol 95% (-20C, 1ml for each 1 x 10 6 cells)
adding the methanol drop by drop mixing gently with vortex.
1. Incubate the cells for 10 min at 20C. After this step, cells can be stored
subsequently for one week at 4oC.
6. Centrifuge the cells for 7 min at 4000 r.p.m
7.
Resuspend the cells in 200-250 l of PBS containing 2% FBS
8. Place the cell suspension to a FACS tube with 2ul of stock PI (1mg/ml) solution in
the bottom. In this step and the follow to mantein on ice and dark.
24
9. Fluorescence emission of samples is analyzed by Flow Cell Cytometer (FACS;

Becton Dickinson, Mountain View, CA) in the FL-3 channel with the Cell Quest
program.
On the FACs from the Lab Inmunobioquimica (Facultad de Cs. Qcas y Farmaceuticas),
the settings for HT-29 cells used in December 2001 were:
Detectors/Amps
Detector Volt
Threshold
Amp gain
Mode
FSH
P1 FSC
E00
1.49
Line
SSC
P2 SSC
249
Log
FL1
P3 FL1
460
Log
FL2
P4 FL2
250
Log
FL3
P5 FL3
385
Log
48
Example of Results:
control cells, 5% serum

M1: hipodyploid population
Vicky Monardes, Jan 2002
25
IN SITU CASPASE-3/CASPASE-8 ASSAY BY FACS

6
1. A20 and Jurkat cells (0.5x10 /ml) were incubated in complete medium with FasL (100
ng/ml) for 4 h at 37C in a total volume of 500 L. For the inhibition experiments,
cells were pre-incubated for 30 minutes with zVAD-fmk (5 M).
2. Subsequently, cells were harvested by centrifugation at 3.000 r.p.m. for 7 min at room
temperature (This step is necessary to eliminate all culture medium and thereby avoid
dilution of the substrate).
3. Resuspend the cell pellet in 15-20 l of each substrate and incubated for 1 h in the
presence of caspase-3 substrate FAM-DEVD-fmk (Promega) or the caspase-8 substrate
FAM-LETD-fmk (Intergen) at 37C and 5% CO2. Every 15 min resuspend the pellet
with care.
4. Add 1 ml of PBS 1x and centrifugate the cells at 3.000 r.p.m. for 7 min. Repeat the
washing step one more time.
5. Resuspend the pellet in 300 l PBS 1x and analyze the fluorescence emission by Flow
Activated Cell Sorter (FACS; Becton Dickinson, Mountain View, CA) using the Cell
Quest program. The subtrates are measured with the FL-1 channel.
For more details see The complete CaspaTag manual, Intergen, caspase activity kit.
www.intergenco.com
Claudio Hetz, Jan 2002
26
VIABILITY ASSAYS BY FACS

-
Seed 100 l of A20 cells (0,5 x 106 cell/ml) in a 96 wells plate.
Add FasL to final concentration of 100 ng/ml and incubate the cells for 16 h at
37C.
Harvest the cells and wash each well with 200 l of PBS plus 2% FCS.
Centrifuge the cells for 7 min at 3.000 r.p.m.
Note: For DNA content analysis:

2. Cells were harvested and the pellet were resuspended vigorously by hand
and then the tubes were put on ice.
3. The cells were resuspended in methanol 95% (-20C, 1ml for each aliquot of
1 x 106 cells) adding the methanol drop by drop mixing gently with a vortex.
4. Incubate the cells for 10 min at 20C. This step allows subsequent storage
of cells for one week.
-
Resuspend the cells in 250 l of PBS containing 2% FCS for FACS analysis and
transfer the cell suspension to a FACS tube.
Stain the dead cells with 10 g/ml of propidium iodide (PI) to determine cell viability.
Samples containing roughly 1x104 cells were analyzed by FACS in the channel FL-3
using the Cell Quest program.
27
ASSESSMENT OF CHROMATIN CONDENSATION AND MORPHOLOGICAL

CHANGES
1. Cells were treated as described previously (viability assays), and stained after 16 h
cells with PI (1 g/ml) for 5 min in PBS on ice.
2.
After washing twice in PBS 1x, cells were treated with glycerol-DABCO and
viewed by an Axiovert 100 Carl Zeiss confocal microscopy upon excitation at
488nm using a 610nm emission filter. As a control, cells were permeabilized by
addition of 500 l ice-cold ethanol and incubated for 10 min at 20C before
staining with PI.
28
QUANTIFICATION OF PHOSPHATIDYLSERINE EXPOSURE BY FACS

Presence of phosphatidylserine in the outer leaflet of the plasma membrane was detected
following instructions of the manufacturer by flow cytometry using FITC-coupled annexin
V (annexin V-FITC) using the ANNEXIN-V-FLUOS STAINING KIT (Roche, 1858777).
1. Seed 100 l of A20 cells (0,5 x 106 cell/ml) in a 96-well plate.
2. Add FasL to final concentration of 100 ng/ml and incubate the cells for 16 h at
37C.
3. Harvest the cells and wash each well with 200 l of PBS plus 2% FCS. Centrifuge
the cells for 7 min at 3.000 r.p.m. (This is necessary to eliminate all culture medium
and thereby avoid dilution of the probe).
4. Resuspend the cells in 25 l of the following solution:
Propidium iodide
Annexin-V-FLUOS
0.5 l
1 l
24 l
Binding buffer
As controls and to calibrate the cytometer (compensation of fluorescence), stain the

negative (non-treated cells) and the positive (i.e. FasL-treated cells) controls with either
Annexin or PI, respectively.
5. Incubate the cells for 30 min at room temperature in darkness. Every 10 min
resuspend the pellet with care.
6. Add 250 ml of binding buffer (at 4 C) and analyze the samples by FACS. AnnexinV-fluos is measured in the FL-1 channel and PI for this experiment in the FL-2
channel.
29
DNA FRAGMENTATION ASSAY
Method
After the respective incubations, cells are washed twice in 400l cold PBS following
centrifugation for 5min at 3000-4000 rpm (Eppendorf centrifuge). To the pellet of washed
cells add 100l of cold PBS and 100l of phenol:chloroform:isoamyl alcohol (25:24:1).
Mix vigorously for 30sec and centrifuge 5min at 13000 rpm and 4oC (if possible). Separate
phases should be visible after centrifugation. If not, repeat this step. Remove very carefully
upper aqueous phase and put in a fresh Eppendorf tube. Mix 17l of this supernatant with
2ml RNAse Buffer (Buffer 3 from Gibco-BRL) and 1l RNAse A (1mg/ml) and incubate
for 30 min at 37oC. Then add 4l of 6x DNA loading buffer, load onto a 2%agarose gel and
run at 70-100V for 15-30min or until the blue dye front migrates 3/4 down the gel. View on
transilluminator with UV source.
Based on protocol by Martin Hunn
Established in Chile by Patricio Rojas, July 2001
30
CASPASE-3 ACTIVITY ASSAY (CHROMOGENIC)

After the respective incubations, cells are washed twice in 400l cold PBS following
centrifugation for 5min at 3000-4000 rpm (Eppendorf centrifuge). Cell pellets are left on
ice until processed further. To cells add 100l of CHAPS buffer containing protease
inhibitors and 1l of the caspase-3 substrate pNA-DEVD (final conc. 100M) and
resuspend by pipetting up and down 5-6x (N.B. solution becomes viscous due to liberation
of DNA). Incubate for 30min at 37oC and then centrifuge 15min at 13000rpm and RT.
Remove supernatant, place in ELISA plate and continue incubation at 37oC for 1h. Read in
ELISA plate reader at 405nm (best reader in Immunology or Parasitology).
Note.
1) use CHAPS buffer with inhibitors and substrate to determine background value.
2) Once cells have been washed with PBS they may be stored as a frozen pellet at 20oC.
Based on protocol by Martin Hunn
31
C: BIOCHEMISTRY
TX-114 EXTRACTION..................................................................................................34
GEL RECIPES................................................................................................................35
DETERMINATION OF PROTEIN CONCENTRATION WITH BCA.........................36
PROTEIN CONCENTRATION DETERMINATION WITH BIO-RAD KIT...............37
CAVEOLAE PURIFICATION FROM MDCK CELLS IN SODIUM CARBONATE. .38
CHLOROFORM:METHANOL PROTEIN PRECIPITATION FOR GEL
ELECTROPHORESIS....................................................................................................40
14
C-CHOLESTEROL LABELING OF CELLS BEFORE FRACTIONATION ON
SUCROSE GRADIENT..................................................................................................41
PREPARATION OF MOUSE COLON CRYPTS...........................................................43
CYTOKERATIN PURIFICATION FROM TISSUES...................................................44
IN GEL MAPK ASSAY (ERK).......................................................................................45
IN GEL MAPK ASSAY (JNK).......................................................................................47
PHOSPHOAMINO ACID ANALYSIS: MARK KAMPS'S METHOD.........................49
POURING GELS............................................................................................................51
AMIDOSCHWARTZ PROTEIN DETERMINATION...................................................52
PETERSON PROTEIN ASSAY.....................................................................................53
SILVER STAIN PROCEDURE BIO-RAD KIT.............................................................54
SUBCELLULAR FRACTIONATION...........................................................................55
EXTRACTION OF TISSUE CULTURE CELLS AND T-CELLS.................................58
T-CELL PREPARATION................................................................................................59
T-CELL LABELING WITH 35S METHIONINE/CYSTEINE.......................................60
TISSUE ISOLATION......................................................................................................61
TISSUE EXTRACTION.................................................................................................62
PREPARATION OF BAL 1000X STOCK.....................................................................63
16-BAC/SDS-PAGE: A TWO-DIMENSIONAL GEL ELECTROPHORESIS..............64
BIOTIN (NHS) LABELLING OF PROTEINS..............................................................67
BRADFORD PROTEIN ASSAY....................................................................................68
STOCK SOLUTIONS FOR RIPA BUFFER..................................................................69
BUFFER TRITON TO WASH IMMUNOPRECIPITATES...........................................70
PBS (PHOSPHATE BUFFER SALINE)........................................................................70
CM (CULTURE MEDIA)...............................................................................................70
OTHER BUFFERS.........................................................................................................71
HEPES BUFFER.............................................................................................................72
TN BUFFER....................................................................................................................72
CHAPS EXTRACTION BUFFER..................................................................................72
32
PREPARATION OF GST-PBD BEADS (FOR CDC42 AND RAC ASSAYS)..............73

RAC1 AND CDC42 ACTIVITY ASSAYS.....................................................................74
STIMULATION OF ....... CELLS (IP AND ENDOF TREATMENT)...........................75
CHOLESTEROL EXPERIMENT: STIMULATION OF EL-4 CELLS AFTER
CHOLESTEROL DEPLETION (ANTI-PY BLOT).......................................................76
GEL SCANNING............................................................................................................78
IODOBEAD PROTEIN IODINATION.......................................................................79
IODOGEN PROTEIN IODINATION.............................................................................80
KINASE BUFFER..........................................................................................................81
LABELLING OF ASTROCYTES MEMBRANES WITH BIOTIN (NHS)..................82
MEASURING ESTERASE ACTIVITY OF ACROSIN.................................................83
SPERM MEMBRANE PREPARATION........................................................................84
MEMBRANE PREPARATION......................................................................................85
PREPARATION OF GST-RBD (FOR RHOA ASSAYS)...............................................86
RHOA ACTIVITY ASSAYS...........................................................................................87
SPERM MEMBRANE PREPARATION........................................................................88
SURFACE LABELLING (NHS-SS-BIOTIN) OF EL-4 CELLS...................................89
SURFACE LABELLING OF ASTROCYTES (DI-TNC1) WITH BIOTIN (NHS)......90
SURFACE LABELLING OF EL-4 CELLS WITH BIOTIN (NHS)..............................91
THY-1 PURIFICATION FROM EL-4GB RIPA EXTRACTS.......................................93
STRIPPING BLOTS.......................................................................................................94
NITRITE MEASUREMENT ASSAY.............................................................................95
ISOLATION OF MITOCHONDRIA..............................................................................97
PURIFICATION OF DIGS.............................................................................................98
PURIFICATION OF LIGHT MEMBRANE FRACTIONS BY TRITON X-100
EXTRACTION IN SUCROSE GRADIENT................................................................101
DOT BLOTTING WITH CHOLERA TOXIN, GM1 DETECTION IN RAFT
FRACTIONS.................................................................................................................105
33
TX-114 EXTRACTION
1. Material
TBS 10X
0.1 M Tris-HCl pH 7.4

10 mM EDTA
1.4 M NaCl
12, 1 and 0.06% TX-114 in TBS (Serva, Nr. 37243)

LAP
CLB
leupeptine
pepstatine
antipain
5 mg/ml (Fluka Nr. 62070)

0.5% Nonidet P-40 (NP-40, Fluka Nr.74385)

0.5% deoxycholic acid (Sigma Nr. D-6750)
25 mM Tris-HCl pH 8.2
50 mM NaCl
0.01% NaN3
2. Protocol
5 x 106 cells are lysed in 1 ml of cold TX-114 + 4.4 l LAP at 4 C (107 cells in 2 ml of the
same mix) inside 2ml Eppendorf tubes on ice for 1 hour. Mix the tubes from time to time.
Eliminate the nucleus by centrifuging the tubes 5 minutes at 3000 RPM in the cold room
(Eppendorf centrifuge).
Rescue the supernatant and warm it for 2 minutes at 32 C.
Then centrifuge 1 minute the tubes at 6500 RPM in an Eppendorf centrifuge placed at RT.
Separate the two phases.
Resuspend the detergent phase (about 50 l) in 1 ml of cold 0.06% TX-114 and add to the
aqueuse phase (about 1 ml) 50 l of cold 12% TX-114.
Warm both detergent and aqueuse phases at 32 C for 2 minutes.
Centrifuge 1 minute the tubes at 6500 RPM
Separate the two phases and pull both detergent respectively soluble fractions together in
the same tube.
3. Reference
1. Bordier, C. (1981). Phase separation of integral membrane proteins in
114 solution. J. Biol. Chem. 256, 1604-1607.
34
Triton-X
GEL RECIPES
(Enough for 2 minigels)
Lower Gel:
H2O
Acryl/Bis
Lower Tris
10% APS
TEMED
4%
6.16
1.33
2.50
0.03
0.01
6%
5.49
2.05
2.50
0.03
0.01
8%
4.82
2.68
2.50
0.03
0.01
10%
4.15
3.35
2.50
0.03
0.01
12%
3.48
4.02
2.50
0.03
0.01
12.5%
3.32
4.18
2.50
0.03
0.01
15%
2.50
5.00
2.50
0.03
0.01
Upper Gel:
H2O
Acryl/Bis
Upper Tris
10% APS
TEMED
3.25
0.50
1.25
0.02
0.01
Solutions:
Acryl/Bis (30%):
29.2 g Acrylamide (Biorad 161-0101, electrophoresis purity),
0.8 g BIS (N, N' methylene-bis-acrylamide, Biorad 161-0200, electrophoresis
purity)
100 ml H2O.
Make in a dark bottle, store 4 C in dark.
Lower Tris: 1.5 M Tris, pH 8.8
0.4% SDS
Upper Tris: 0.5 M Tris, pH 6.8
0.4%SDS
10% APS: Ammonium persulfate, electrophoresis grade. Either make fresh just before
use or store frozen in tiny aliquots (100-150 l) and never re-use.
TEMED: Biorad 161-0800. Store 4 C, keep tightly covered and on ice when using.
RUNNING BUFFER 10x: 30g Tris (0.25 M), 174g glycine (2.32M) per litre. Final pH
after dilution 8.3. For electrophoresis add 10ml 10% SDS per litre of chamber
buffer
SAMPLE BUFFER 2x: 10ml glycerol 87%, 5ml beta-mercaptoethanol, 30ml 10% SDS,
12.5ml Upper-Tris per 100ml final volume. Add spatula tip of Bromphenolblue and
shake well to dissolve.
35
DETERMINATION OF PROTEIN CONCENTRATION WITH BCA

Features
- linear working range for BSA from 20-2000 g/ml
- compatible with most ionic and nonionic detergents
- interfering substances
100 mM EDTA
1mM DTT
20 % ammonium sulfate
Material and Solutions
BCA Protein Assay Reagent (Pierce N 23225)
Reagent A: Sodium carbonate and bicarbonate, BCA
and sodium tartrate in 0.1 M NaOH
Reagent B: 4% CuSO4 5H2O
Albumine Standard: 2 mg/ml
detection reagent
Microtiter plates (Dynatech Lab. N 001-010-2801 are the cheapest)

Method
Mix 200 l of reagent B with 10 ml of reagent A. This A+B solution is stable for 24 hours.
Follow the indication of the table to obtain appropriate BSA dilutions to establish a
standard curve.
Table 1: Preparation of the diluted BSA standard
Vol. of the BSA to add
300 l of (Stock) 0 l
175 l of (A)
325 l of (B)
325 l of (D)
325 l of (E)
100 l of (F)
Vol. of diluent to add

2000 g/ml
1500 g/ml (A)
1000 g/ml (B)
175 l
325 l
325 l
325 l
400 l
Final BSA Conc.
750 g/ml (C)

500 g/ml (D)
250 g/ml (E)
125 g/ml(F)
25 l g/ml (G)
Then mix, at least in duplicate, directly in the ELISA plate, 10 l of each BSA dilution or
10 l of the sample to be analysed (make two to three different dilutions) with 200 l of
solution A+B.
Wrap the plate with parafilm and incubate it 30 minutes at 37C (GP's lab).
Read OD at 562 nm.
36
PROTEIN CONCENTRATION DETERMINATION WITH BIO-RAD KIT

To prepare
Standard curve
In Eppendorff tubes pipette the following volumes in l:
BSA 1.4 mg/ml 0
H2O
400
2
398
4
396
6
394
8
392
10
390
15
385
20
380
30
370
7.5
10
15
Final concentrations:
g/ml proteins
Samples
Prepare different dilutions of the samples to be analysed in Eppendorf tubes to a final
volume of 400 l.
Reaction
Add to all samples 100 l of 5x of Bio-Rad reagent, vortex and leave for 15 minutes at RT.
Transfert 200 l of all reactions in an ELISA plate and read OD at 620 nm.
37
CAVEOLAE PURIFICATION FROM MDCK CELLS IN SODIUM CARBONATE

Material and solutions
confluent MDCK cells in 2 plates of 150 mm (50-100.106 cells)
glass loose-fitting Dounce homogenizer B (cell culture shelf, annoted MR )
Polytron tissue grinder (JP Kraehenbul's lab)
Sonicator (5 floor)
Beckmann centrifuge tubes 14x95 mm
4 ml poly-propylen tubes (Milan)
rubber policeman
PBS
500 mM Sodium Carbonate pH 11
5.3 g Na2CO3
H2O to 100 ml
(adjust pH with HCl 10 N)
MBS pH 6.5
25 mM Mes (Sigma)
150 mM NaCl
6.25 ml 1 M
7.5 ml 5 M
H2O to 250 ml (adjust pH
Benzamidine
Antipain
Leupetin
20 mg/ml
4 mg/ml
0.25 mg/ml
with NaOH 10N)

BAL 1000x
90% sucrose
35% and 5% sucrose
in MBS
in MBS containing 250mM Na2CO3
solubilise the sucrose solution in 50 ml Falcon tubes by boiling

either in a water-bath or simply heating in a micro-wave oven
38
solutions
Method
Cell lysis:
All steps are done at 4C on ice.
Wash cells twice with cold PBS. Eliminate carefully all PBS after the last wash.
Add 1ml of cold Na2Co3 + 2l of BAL per plate and detach the cells using the rubber
policeman. Pool cells from the two plates into a 15 ml Falcon tube. Homogenisation is
carried sequencially in the following order using:
1) a loose-fitting Dounce homogenizer. Homogenize extremely carefully
with 10
strokes. Be careful:
1 to avoid making foam by never pulling the piston out of the
liquid phase
2 to move the piston very slowly (10 sec per stroke)
2) a polytron tissue grinder: three 10 sec burst, 2.5 V
3) a sonicator: three 20 sec bursts, 2.5 Watt
Gradient preparation:
Using graduated pipetes, place 2 ml of 90% sucrose at the bottom of a Beckman tube, 4 ml
of 35% and 5% sucrose in 2 Milan tubes. Note that these solutions are extremely viscous so
pipeting requires a lot of precision. Keep these solutions at room temperature.
Mix exactly 2 ml of the lysate with the 2 ml of 90% sucrose in the centrifugation tube. Use
a blue tip with a severed end to enlarge the aperture through which liquid are displaced
upon pipetting. Pipet up and down delicately the mix until cell lysate and 90% sucrose are
well mixed. Then put the tube on ice to cool it to 4C.
A discontinous gradient is formed above the homogenate by overlaying delicately first 4 ml
of the 35% and then 5% sucrose solution. For that, use a Pasteur pipet and add, drop after
drop, the sucrose solution on the surface of the tube.
Introduce the centrifugation tubes into the SW40-Ti bucklet which is then fixed on the
rotor. Insert all the bucklets on the rotor, balance them and start the centrifugation using the
foolowing parameters:
- brake off
- rotor code 3
- speed 30'000 rpm
- 4C
- time 20 hrs
Fraction collection:
After centrifugation, insoluble membrane fractions are flotating round the 35 and 5%
interphase. A large amount of material is also visble at higher sucrose density. An small
insoluble pellet is attached to the bottom of the tube.
Collect 1 ml fractions starting from the top of the tube with a 1 ml seringe. Try also to
collecte the pelet, by vortexing it in 1 ml of Na2CO3. In general, analysis of 10 l from each
fraction collected is sufficient to detect the enrichment of caveolin-1 in fraction 4 and 5 by
SDS-PAGE and Western Blotting.
Reference
1. Song et al. J. biol. Chem. 1996, 271: 9690-9697
39
CHLOROFORM:METHANOL PROTEIN PRECIPITATION FOR GEL

ELECTROPHORESIS
Nov. 1991
Wessel and Fluegge (1984) Anal. Biochem. 138:141-143.
For up to 400 l sample in 1.5 ml polypropylene tube:
Add
400 l methanol
100 l chloroform Mix
q.s. water (to total aqueous vol of 400 l) Mix
Spin 5 min
There will be two phases, with chloroform on the bottom and the protein at the interface.
Remove the supernatant to near the interface but do not remove any of the interface.
Add 300 l methanol. Mix. Spin 5 min.
Carefully remove the entire sup (use a pasteur pipet with a drawn tip).
Place the tubes under vacuum for 10 min to remove the last traces of solvent.
Resuspend the pellet in equal volumes water and 2x sample buffer for gel electrophoresis.
40
14
C-CHOLESTEROL LABELING OF CELLS BEFORE FRACTIONATION ON

SUCROSE GRADIENT

adherent or non-adherent cells (MDCK and EL-4 as representative exemples)
[4-14C]-cholesterol
Du Pont NEC-018
0.040Ci/l
in ethanol
Pyrex glass tube 8 ml with corresponding lid

Hamilton syringe
a special one is stocked in Andrew's drawer
100% ethanol
Complete cell culture medium (containing serum)
Method
Labeling medium 6-12 hours preincubation
Under sterile hood.
Rinse Pyrex tube, lid and Hamilton syringe with ethanol before pipetting the cholesterol
solution (10l or 0.4Ci/set of cells) into the Pyrex tube. Rinse the Hamilton syringe once
with 30-40l of cold ethanol and pool this wash ethanol together with the cholesterol into
the Pyrex tube. Then rinse several times the syringe in a 5ml plastic tube countaining
ethanol, which is eliminated afterwards with liquide radioactive waste.
Out of the hood but work as sterile as possible.
Dry the cholesterol solution under nitrogen (Valituti's lab) by introducing directly inside the
Pyrex tube a Pasteur pipette connected to the nitrogen source. It might take 10-15 minutes.
Sterilize the Pasteur pipette with ethanol before use.
Under the hood again.
Fill completely the Pyrex tube with the appropriate serum (8-9ml) containing medium
(minimize air volume that could oxydize medium and thus cholesterol). Close the tube with
the lid and incubate the labeling solution 6-12 hours at 37 C in the incubator.
Cell labeling: 36-48 hours
First, estimate the number of cells to start the experiment knowing that after the backextraction procedure the total cell number should represent around 10mg of total proteins (2
confluent 15 cm dishes of MDCK cells or 100-130 x 106 EL-4 cells.
MDCK: 20-25% confluent cells in 2x15cm dishes (split a confluent 10 cm dish the evening
before the experiment). Wash once with PBS and add the labeling mix to the cells together
with 20 ml of cold medium per plate (Final concentration: 8nCi/ml)
EL-4: 15 x 106 cells. Resuspend cell pellet with 40 ml of medium + the labeling mix (Final
concentration: 8nCi/ml)
41
Leave the cells to grow in incubator for 48 hours.

Back-extraction: 6-12 hours
MDCK: Suck medium into radioactive waste and wash cells once with PBS. Add fresh cold
medium for a 6-12 hours incubation.
EL4: After pelleting the cells continue as for MDCK.
Next day, wash cells and proceed for DIG's isolation
Schedule example
Friday morning
Friday evening
Sunday evening
Monday morning
Preparation of labeling solution

Incubation up to the evening (10-12 hours)
Addition of labeling solution to the cells
Cell labeling 48 hours
Washing of cells
Back-extraction (12 hours)
DIG's isolation or other experiment
Radioprotection
The radioactivity used is very low but some precautions should be taken.
1. Protect the working areas.
2. Labeling solutions together with the ethanol used to wash the
Hamilton syringe
are collected in a bottle for radioactive waste.
3. All plastic and biological material which may contain radioactive traces that can
be disposed in the normal waste but should first first, sealed in a plastic bag.
Comment
Depending on the method of preparation to isolate caveolae-like domains, the cholesterol
distribution after external labelling may vary. Using the sodium carbonate method most
(80%) [14C]-cholesterol is recovered in the light fraction, while with the detergent method,
recovery in this fraction is 40%.
42
PREPARATION OF MOUSE COLON CRYPTS

1
Take colon from mouse and place immediately in PBS.
Cut open the colon longitudinally and rinse out faeces with PBS.
If intending to culture the crypt cells, place colon(s) in a solution of 0.04% sodium
hypochlorite (= ~1% commercial bleach) in PBS for 20 mins at R.T. If not
culturing, this step can be omitted.
Place colon(s) in 10 ml sterile digestion mix (sterile if intend to culture cells

afterwards) comprising 3 mM EDTA (111.6 mg) and 0.5 mM DTT (7.7 mg) in 100
ml PBS, for 60-90 mins at R.T or 4oC.
Remove colon(s) from digestion mix and rinse gently once or twice with a total of
20 ml PBS.
Place 10 ml PBS in 30 ml tube with colon(s). Cap the tube and shake lightly for a
couple of seconds. Draw off PBS and transfer to 10 ml tube marked "1".
Repeat step"6" but this time shake the tube VIGOROUSLY for about 5 seconds.
Transfer to tube marked "2". This will contain an almost pure preparation of intact
epithelial crypts - take a drop and check in the microscope
Step "7" can be repeated to release more epithelial cells, but the crypts may be
broken up at this stage (which doesnt matter for an RNA prep. so long as the there
is not significant stromal contamination).
Spin tubes 1, 2 & 3 at 400 rpm with brake, for 5 min at 4oC.
10
Tip off PBS and resuspend crypts as appropriate for your needs.
This procedure can also be followed to release crypts from human biopsy material.
(Human crypts are longer than those from the mouse).
43
CYTOKERATIN PURIFICATION FROM TISSUES

(Modification of protocol from E. Reichmann/W. Franke for culture cells)
Make buffers the day before and store at 4C. The amounts of components indicated is for a
buffer volume of 500ml.
Tris
NaCl
Triton-X100
EDTA
KCl
Low Salt Buffer

0.6
g
9.08 g
5
g
0.93 g
High Salt Buffer

0.6
g
9.08 g
5
g
0.93 g
55.9 g
pH to 7.6 with NaOH (10M) - otherwise EDTA does not dissolve.

Cut tissue into small pieces with a fine pair of scissors. Add 10-fold excess over tissue
weight of low-salt buffer, together with PMSF and Benzamidine.
Sonicate 3x10'' with probe sonicator at power level 2-3.
Dounce homogenize 10x - sonicate again 15" at power level 2-3.
Spin in clinical centrifuge at 5000 rpm, 4C for 5 min.
Homogenize again briefly with the Dounce homogenizer 5x in the presence of high-salt
buffer. Volume might have to be doubled here, since in some cases solution will now
become very viscous.
Spin at 500 rpm, 5' to remove collagenous material. Particularly in the case of tissues like
lung, this might be necessary.
Extract on a rotating shaker for 30 min. or more at 4C.
Collect cytokeratins by centrifugation for 30 min. at 5000 rpm, 4C in clinical centrifuge.
Carefully remove supernatant,
preferably with a pasteur pipette to avoid moving
weakly attached pellet.
Resuspend cytokeratin pellet from about 1-1.5g of tissue in 1 ml of PBS and split up
between 10 eppendorf tubes. Spin again 5 min. at 13000 rpm, remove supernatant fluid and
freeze pellets at -20C.
Each pellet, when completely solubilized in sample buffer should yield enough protein for
2-4 lanes with roughly 10-20 ug of cytokeratin per lane. Two major bands of 53 to 55 kDa
are visible above the actin contamination present around 50 kDa.
(N.B. cytokeratin pellets from lung are not easily solubilized even in sample buffer, while
the corresponding proteins from brain are).
44
IN GEL MAPK ASSAY (ERK)

Protocol from Felipe Sierra (developed for Splenocytes)
1. Cell Stimulation
- wash cells in 1x serum-free RPMI medium
- resuspend cells at 5x106 cells/ml in serum-free medium and aliquot 3ml into
each plate
- equilibrate at 37C, 5% CO2 for 1-2 hours
- add 5% fetal bovine serum and stimulate, e.g. with PMA (50 ng/ml) plus A23187
(1ug/ml)
2. Whole Cell Extract Preparations
- resuspend cells in 100ul lysis buffer (15x106 cells/sample)
- shake the cell suspension at 4C for 30 min
- clarify by centrifugation at 12000xg for 10 min
- collect the supernatants (WCE)
- measure protein by the Bradford method (BioRad)
3. Preparation of gel containing myelin basic protein (MBP)
Separating gel (12.5%)
ddH2O
1.5ml
1.5M Tris pH 8.8/ 4mM EDTA
1ml
MBP (10mg/ml in pre-kinase buffer
200ul
(final conc. 0.5 ug/ml)
10% SDS
40ul
40% acrylamide/bis (29:1)
1.25ml
10% APS
20ul
TEMED
4ul
Stacking gel
the same as for a normal SDS-PAGE gel
4. Electrophoresis
- load reduced samples as usual (10ug/sample) along with pre-stained markers
- run at 200V for about 60-80 min (until prestained markers indicate that your
kinase of interest is in the middle of the gel. N.B. proteins run slower than in normal gels
5. Removal of SDS
- wash gel 3x in 100ml propanol buffer at room temperature (20 min each)
- equilibrate for 1hr in 100ml ERK buffer with continuous agitation
6. Denaturation
- incubate gel for 1hr at RT with continuous agitation in 100ml denaturing buffer
7. Renaturation
- incubate the gel in 100ml renaturing buffer with gentle agitation overnight at 4C.
- the next day, wash at least another 3x in 100ml of the same buffer
8. Kinase Assay
- equilibrate the gel in 15ml pre-kinase buffer with gentle agitation for 1hr RT
- the kinase assay is done by incubating the gel for 1hr at RT in 15ml kinase
buffer containing 20mM ATP and 100uCi (32P-ATP)
9. Detection
- wash the gel in 100ml wash buffer at RT 30min-1hr each time, then leave it
washing overnight
- the next day, continue washing at least 3 times in 100ml of the same buffer
- autoradiography as usual
10. Solutions
Lysis buffer
25 mM Hepes pH 7.7
45
0.3 M NaCl
1.5 mM MgCl2
0.2 mM EDTA
0.1% Triton X-100
before use add:
2mM NaPPi
0.5mM DTT
20 mM b-glycerophosphate
5 ug/ml leupeptin
10 ug/ml aprotinin
100 ug/ml PMSF
0.1 mM sodium orthovanadate
Propanol buffer
20% 2-propanol in 50 mM Tris-HCl pH 8.0
ERK buffer
5 mM DTT
Denaturing buffer
6M guanidine-HCl in ERK buffer
Renaturing buffer
ERK buffer containing 0.04% Tween 20, 2mM EDTA
Pre-kinase buffer
20 mM Hepes pH 7.6
20 mM MgCl2
2 mM DTT
5 mM b-glycerophosphate
0.1 mM Na3VO4
Wash buffer
5% (w/v) trichloroacetic acid
1% NaPPi
AQ, June 98
46
IN GEL MAPK ASSAY (JNK)

Protocol from Felipe Sierra (developed for Splenocytes)
1. Cell Stimulation
as for ERK assay
2. Whole Cell Extract Preparations
as for ERK assay
3. Preparation of gel containing myelin basic protein (MBP)
Separating gel (12.5%)
0.75 mm
ddH2O
1.5M Tris pH 8.8/ 4mM EDTA
GST c-Jun to final conc of 60 ug/ml
10% SDS
40% acrylamide/bis (29:1)
10% APS
TEMED
$
1mm comb
1.65ml 2.15ml
1ml
1.25ml
x ml
x ml
*40ul
*50ul
1.25ml 1.55ml
20ul
25ul
4ul
5ul
(* since the eluate solution for GST c-Jun contains SDS, make sure the final
concentration of SDS is about 0.1%. The indicated values are correct if no
additional SDS is present)
Stacking gel
the same as for a normal SDS-PAGE gel
4. Electrophoresis
same as for ERK, but load 30ug/sample
5. Removal of SDS
- wash gel 2x in 100ml propanol buffer at RT with continuous agitation (20 min
each time)
- equilibrate for 2x in 100ml Jnk buffer at RT with continuous agitation
(20 min
each time)
6. Denaturation
- incubate gel 2x in 100ml denaturing buffer with continuous agitation (20 min
each time)
7. Renaturation
- discard half of last volume (50ml), add 50ml renaturing buffer, continue shaking
for 15min
- repeat this dilution step 3 or 4 times
- wash the gel with 100ml renaturing buffer overnight at 4C
- the next day, wash again once in 100ml of the same buffer
47
8. Kinase Assay
- equilibrate gel in 15ml pre-kinase buffer with gentle agitation for 30 min at
4C
- the kinase assay is done by incubating the gel for 2hr at 30C in 15ml kinase
buffer containing 20mM ATP and 100uCi (32P-ATP)
9. Detection
- same as for ERK
10. Solutions
Lysis buffer
same as for ERK
Propanol buffer
20% 2-propanol in 50 mM Hepes pH 7.6
JNK buffer
50 mM Hepes pH 7.6
5 mM b-mercaptoethanol
Denaturing buffer
6M urea in JNK buffer
Renaturing buffer
JNK buffer containing 0.05% Tween 20
Pre-kinase buffer
same as for ERK
Wash buffer
same as for ERK
AQ, June 98
48
PHOSPHOAMINO ACID ANALYSIS: MARK KAMPS'S METHOD

...Recipes for the buffers are at the end of this protocol.
1. Label your protein with 32Pi. Then subject the protein to SDS polyacrylamide gel
electrophoresis and transfer your gel-fractionated protein to Immobilon-P.
Neither nitrocellulose nor nylon will work!
Keep the membrane wet and wrap in Saran wrap.
Add radioactive markings for orientation of film later
Expose to film.
2. Cut out band of interest, re-wet in methanol, rinse well in water and place in a screw-cap
tube containing 150 ul 5.7 N HCl or enough to submerge your piece of membrane.
3. Incubate in 110C oven for 60 min.
4. Microfuge sample full speed for at least 1 min.
5. Transfer supernatant to new tube and lyophilize on Speedivac. (It takes about 3 hr.)
6. Resuspend in H2O, and microfuge 5 min. Spotting more than 3 ul is tedious, so don't use
much H20. On the other hand, I wouldn't use much less than 6 ul so as to have good
recovery.
7. Spot 1 ul PAA standards (about 0.3 ug each of PSer, PThr, and PTyr) on a cellulose thin
layer chromatography plate and then spot your sample. We use 0.1 mm EM cellulose plates
You can spot the whole sample if you are skillful and have no choice because you don't
have many counts.
Spot 0.25-0.30 ul at a time and dry with house air, blown through a plugged pipet,
between applications.
How do I wet the plate? We use a blotter that has four, 2 cm, circular holes cut out of it,
one for each origin. We cut the holes with a sharp cork borer. The blotter can be made from
a good grade of blotting paper, or two layers of Whatmann 3MM sewn together. It should
be wet, but not dripping. For the first dimension, you wet the plate with pH 1.9 buffer.
8. Electrophorese at pH 1.9, 1.5 KV, 20 min in the first dimension.
9. Let plate air dry well.
How do I wet the plate for the second dimension? Use three rectangular pieces of
Whatmann 3MM that have been wet with pH 3.5 buffer. Wet the bottom of the plate below
the lower two origins. Keep the paper at least 1 cm away from where the PAAs are. Then
wet the area between the 4 origins. Finally wet the top of the plate. The blotters should be
quite damp, but not soaking wet.
10. Rotate 90 counter clockwise. Electrophorese at pH 3.5, 1.6 KV, 13 min in the second
dimension.
The above electrophoresis times are appropriate when you are using a Salk/TVL/MBVL
type electrophoresis rig. A knock off of this is sold by CBS scientific. If you are using
another rig, you will have to optimize your electrophoresis times.
11. Dry plate in oven.
12. Spray plate with ninhydrin sol'n for several seconds. Incubate in oven until can see
purple spots of PAA standards. 15 min should be plenty.
49
13. Mark the plate with radioactive ink so that you can extrapolate where the origins were
and can align the film unambigously with the plate and the PAA markers.
14. Expose the plate to flashed film with a screen a -70C. Film is much preferable to the
phosphorimager because it is transparent and is exactly the same size as the plate and this
facilitates alignment of the film and the plate. In the rare case that you need quantification,
you can use the phosphorimager.
15. After developing the film, trace the locations of the PAA standards and the radioactive
ink marks onto a Xerox transpancy and save this in your notebook.
Recipes
pH 1.9 buffer.
88% Formic acid 50 ml
Acetic acid 156 ml
H2O 1794 ml
Don't use the 98% formic acid and don't adjust the pH.
pH 3.5 buffer
Pyridine 10 ml
Acetic acid 100 ml
H2O 1890 ml
(0.5 mmolar EDTA)
Don't bother to adjust the pH.
There is occasionally a problem with badly smeared PAAs during electrophoresis at pH
3.5. Something seems to leach out of the wicks and make the PAAs relatively insoluble.
Tony thinks it is aluminum. Bart thinks that it's calcium. In any case, this problem can be
prevented by including 0.5 mM EDTA in the 3.5 buffer.
AQ, July 1998
(from kinase database)
50
POURING GELS
September 1993
Clean glass plates, 1 large and 1 small per gel needed. Dry carefully.
In front of casting stand, line up sandwich with large plate next to plexiglass support, 2
spacers, and small glass in front. Tighten sandwich.
Mix in a 15 ml orange cap tube (for a 10 % gel, see gel recipes for other percentage gel
volumes):
2.5 ml lower Tris, cold
4.15 ml H20
3.35 ml Acrylamide/Bis (29:1) (keep cold and dark)
30 l 10% ammonium persulfate (make small aliquots and store frozen, never
reuse)
10 l TEMED (stored in refrigerator)
Mix gently and do not allow bubbles (oxygen inhibits the polymerization reaction; if there
is a problem with the reaction going it is possible to add riboflavin, but it is not usually
necessary for minigels. If used make into 100 l aliquots and store frozen.)
Overlay the gel with water-saturated butanol to keep the interface flat and to prevent air
from inhibiting the reaction.
Remove the overlay by tipping the casting stand and removing the butanol with a pasteur
pipet.
To make the upper gel, mix in another 15 ml conical tube:
1.5 ml upper Tris, cold
3.25 ml H20
0.5 ml Acrylamide/Bis (29:1)
20 l 10% ammonium persulfate
10 l TEMED
Mix as above, fill to the top of the sandwich. Gently insert clean comb until it reaches the
top of the 2 spacers, check and then insert until the teeth are completely sealed.
51
AMIDOSCHWARTZ PROTEIN DETERMINATION

May 1, 1991
Procedure:
1. Put protein sample in a total volume of 270 l in 1.5 ml eppendorf tube.
2. Add 30 l sample solution.
3. Add 60 l 60% TCA. Vortex. Incubate at RT at least 2 min.
4. Set up filter apparatus, using a vacuum flask and millipore 60 mm 0.45 m type
HA filter (mark one side with a soft pencil for orientation of spots later!) on
a fritted glass filter support. With the vacuum on, dot the protein solution
onto the filter using a P-200 pipetman to apply it in a small spot. The liquid
will spread to a much wider area than the spot will if you are not careful.
You can get 9 spots per filter.
5. Rinse the entire filter with about 4 ml of 6% TCA.
6. Immerse the entire filter in amidoschwartz staining solution. Try to keep the
staining time constant between filters, 1 min is good.
7. Rinse for 30 sec in running dH20. (This step may be omitted)
8. Wash x 3 in 20 ml destain, or until the membrane background is white.
9. Dry on a paper towel, with the blue spots up.
10. Using a cork borer cut out the spots and place in 12x75 mm glass tubes.
11. Add 600 l of eluant solution.
12. Incubate10 min RT with 3x vortex. This may be speeded up by placing the
tubes into a 60 C water bath for about 3 min.
13. Read at A 630 ( up to 2 hr after incubation).
The standard curve consists of 1-10 g of protein, the stock protein is 100 g/ ml
BSA.
Solutions:
Sample solution:
1 M Tris-Cl, pH 7.5, 1% SDS
60% TCA
6% TCA
Amidoschwartz staining solution:
0.1% amidoschwartz 10B (napthol blue black - Sigma N3005)in
methanol:acetic acid:H20 (45:10:45 by vol)
Destain:
Methanol:acetic acid:H20 (90:2:8 by vol)
Eluant solution:
25mM NaOH, 0.05 mM EDTA, in 50 vol % EtOH:
1 ml 1 N NaOH
25 l 100 mM EDTA
25 ml 100 % ethanol
23.97 ml H2O
Standards:
100 g/ml BSA in water.
52
PETERSON PROTEIN ASSAY

Using Pierce Micro-BCA Protein Reagent Kit
Nov. 13, 1991
Pipet protein solution into a suitable tube (glass or plastic, 12x75 or 13x100) and bring
volume to 500 l with water.
Add 50 l 0.15% deoxycholate. Mix well. Allow to sit at RT for 10-30 min.
Add 50 l 72% TCA. Mix well.
Centrifuge for 30 min at 2000 rpm in the swinging bucket centrifuge.
At this time make the BCA reagent (mix in order!):
2 parts micro-BCA Reagent C
48 parts micro-BCA Reagent B
50 parts micro-BCA Reagent A
After the centrifuge run, decant the sup and remove any excess liquid.
Add 1 ml BCA reagent and mix.
Incubate at 60 C for 1 hr.
Measure absorbance at 562 nm.
Standard dilutions
Stock 1 = Pierce 2 mg/ml BSA standard
l Stock 1
10
25
50
75
100
Standard
100 g/ml
250
500
750
1000
750
1000
10 l
l Diluent
190
175
150
125
100
[BSA]
100 g/ml
250
500
750
1000
Volume
990 l
10
10
10
Diluent
1.0 g/ml
990
990
990
10
990
20
20
Final [BSA]
2.5
5.0
7.5
10.0
980
980
15.0
20.0
53
SILVER STAIN PROCEDURE BIO-RAD KIT

Revised 11/91
Solutions (for 1-2 10 cm gels)
Fixative 1--40% methanol/10% acetic acid v/v
80 ml MeOH
20 ml glacial acetic acid
100 ml dH2O
200 ml
Fixative 2--10% ethanol/5% acetic acid v/v

42.1 ml 95% EtOH
20 ml glacial acetic acid
337.9 ml dH2O
400 ml
Oxidizer--dilute concentrate 1/10 with dH2O
100 ml
Silver Reagent--dilute concentrate 1/10 with dH2O
100 ml
Developer--Dissolve 32 gm in 1 l dH2O
200-300 ml
Stop solution--5% acetic acid v/v
200 ml
Procedure:
1. Fixative 1
2. Fixative 2
3. Fixative 2
4. Oxidizer
5. dH2O
6. dH2O
7. Silver reagent
8. dH2O
9. Developer
10. Developer
11. Developer
12. Stop
200 ml
200 ml
200 ml
100 ml
200 ml
200 ml
100 ml
200 ml
100 ml
100 ml
100 ml
200 ml
30 min (can be stored here)

15 min
15 min
5 min
5 min
5 min
20 min
1 min
< 1 min (until turns brown)
5 min
5 min (if needed)
5 min
Notes:
1 -- Always wear gloves when handling gels.
2 -- All staining is done in glass or ceramic containers.
54
SUBCELLULAR FRACTIONATION
IMPORTANT: All steps are performed at 4C (centrifugation) or on ice. Buffers are icecold and contain protease inhibitors.
The fractionation experiment is done with Swiss 3T3 fibroblasts, which are 70% confluent.
Per condition, 6 dishes of 15 cm are needed.
Buffers:
Buffer H
25 mMTris-HCl, pH 7.4
2 mM
EDTA
10%
glycerol
+ protease inhibitor cocktail
Nuclear buffer
20 mMTris-HCl, pH 7.4
3mM
MgCl2
+ protease inhibitor cocktail
Protease inhibitor cocktail(PI)

1x BAL (bezamidine, antipain, leupeptin)
4 mM
PMSF
5 mM
DTT
2 mg/ml
pepstatin
1 mM
sodium pervanadate
Preparation of BAL 1000x stock:
Leupeptin stock (Boehringer, 1017101): To 5 mg add 2 ml dd water (conc. 2.5 mg/ml)
Antipain stock (Boehringer, 1004646): To 10 mg add 1 ml dd water (conc. 10 mg/ml)
Benzamidine: To 100 mg add 1 ml dd water (conc. 100mg/ml)
The leupeptin and antipain stocks can be stored frozen at -20C. Do not freeze-thaw more
than about 2x.
Mix:
220l benzamidine
880 l leupeptin stock
440 l antipain stock
760 l dd water
This mix is equivalent to BAL 1000x. Aliquot the mix into 40 Eppendorf tubes with 50-55
l per tube. Store frozen at -20C. Thaw these aliquots only once.
Procedure
Remove medium without scratching the cell monolayer by suction from the side.
Wash cells 2x with 10 ml ice-cold PBS. Be careful, do not disturb cell monolayer (loss of
cells)! Remove the PBS each time.
Add another 5 ml PBS and detach cells with rubber policeman. The cell suspension from
six plates is transferred to a 50 ml NUNC tube.
Centrifuge the cells for 5 min at 1500 rpm (250x g). Remove supernatant.
Resuspend the cells in 5 ml buffer H, spin down the cells, and remove supernatant.
55
Resuspend cells in 1.5 ml buffer H, containing protease inhibitors. The volume of the added
buffer should be about 4x as much as the volume of the cell pellet. Incubate for 10 min on
ice.
The cell suspension is transferred to a B pestle dounce homogenizer. The cells are
homogenized by 30 pestle strokes. Transfer the total cell homogenate into 1.5 ml
Eppendorf tube and put 30 l (2% of total) into separate 1.5 ml Eppendorf tube for analysis
via SDS-PAGE. Test for homogenization efficiency under light microscope with Trypan
Blue.
The cell homogenate is spun for 5 min at 3000 rpm (500x g). This centrifugation permits
separation of the relatively heavy nuclei from the rest of the cell. The supernatant contains
crude cytosol and is transferred to Beckmann centrifugation tubes (approx. 4 ml). Put 30 l
of the crude cytosol aside in separate Eppendorf tube for later analysis. The pellet contains
the intact nuclei and is kept on ice.
The next step requires centrifugation at very high speed in an ultracentrifuge. Note that the
tubes must be precisely balanced! The crude cytosol is spun for 1 hour at 30'000 rpm
(100'000 x g). This centrifugation permits separation of high-molecular membrane
structures from soluble proteins found in the cytoplasm. The supernatant, which contains
the cytosol, is placed in a fresh Eppendorf tube. The pellet contains the total plasma
membrane fraction.
The plasma membrane pellet is washed once with buffer H. Note, be careful not to displace
pellet! Remove buffer.
Then the plasma membrane pellet is resuspended in 1.4 ml buffer H containing 1% Triton
X-100. Incubate on ice for 1 hour.
Resuspend once more and put 30 l aside in separate tube for later analysis. Then the tubes
are centrifuged for 30 min at 30'000 rpm (100'000x g); do not forget to equilibrate tubes
before spin.
Remove the supernatant, which contains the detergent-soluble plasma membrane. The
pellet contains the detergent-insoluble plasma membrane.
The detergent-insoluble plasma membrane fraction are resuspended in 1.4 ml 1x sample
buffer containing reducing agent (DTT).
Intact nuclei are incubated in 2x PCV nuclear buffer containing 0.05% Triton X-100 for 10
min on ice.
Nuclei are transferred to a L pestle Dounce homogenizer. The nuclei are homogenized by
10 pestle strokes.
Transfer the total nuclear extract into 1.5 ml Eppendorf tube and put 2% of total into
separate 1.5 ml Eppendorf tube for analysis via SDS-PAGE.
The nuclear extract is spun for 5 min at 3000 rpm (500x g). The supernatant contains
cellular membranes and transferred into a Eppendorf tube for subsequent analysis.
The pellet containing nuclei is resuspended in nuclear buffer (2x pellet volume) and is
centrifuged for 30 min at 5000 rpm (1900x g) on 45% sucrose.
56
The nuclei pellet is resuspended in buffer H containing 1% Triton X-100 for 1 hour on ice.
Then, it is spun for 30 min at 30'000 rpm (100'000x g).
Transfer the supernatant containing nuclear extract to an Eppendorf tube. Nuclear
membranes in the pellet are resuspended in 1.4 ml 1x sample buffer containing reducing
agent (DTT).
Controls for quality:
Lactate deshydrogenase (LDH):
LDH is a cytosolic protein. It is possible to measure the activity of LDH by following the
decrease of NADH.
Pyruvate
NADH + H+ ------>
L-lactate
The optic density of NADH is measured at 340 nm.

Na-K ATPase assay:
This is a plasma membrane enzyme.
Proteins expected in fractions:
Total cell homogenate:
1592 g
100%
intact nuclei:
120 g
8%
cytosol:
596 g
40%
total plasma membrane:

detergent-soluble plasma membrane:
120 g
82 g
7.5%
5%
detergent-insoluble plasma membrane:
56 g
3%
57
NAD+
EXTRACTION OF TISSUE CULTURE CELLS AND T-CELLS

The adherent cells from a large plate (about 107 cells) are washed briefly with cold PBS +
protease inhibitors (BAL 1x, PMSF 1x), then scraped from the plate surface with a rubber
policeman and washed again 3x in ice cold PBS with protease inhibitors. Basically the
same protocol is used for T-cells except that one starts with ConA blasts, that are first spun
on to a Ficol cushion to separate dead cells (about 107 cells). At this point the cell pellet
may be frozen away at -70C.
To extract the cells add to each pellet 1 ml cold extraction buffer containing protease
inhibitors (1x PMSF, 1x BAL). Homogenise in a Dounce homogenizer with 8-10 strokes at
4C
Sonicate for 2 min. (power input level 1-2, keep sample on ice)
Add Triton X100 (Pierce Surfact-Amps #28314) to final concentration
leave on ice for 20-30 min.
0.1%, vortex and
Spin in Eppendorf cenrifuge at 4C for 30 min. Separate supernatant

from pellet.
Resuspend pellet in an equal volume of extraction buffer containing protease inhibitor
cocktail, vortex
Make aliquots of supernatant and pellet fractions and freeze away at -70C. Do an Amido
Schwarz protein determination. For Overlay assays use at least 100-150 ug of total protein
per lane. For immunoblots amount may vary depending upon antigen. In the case of
caveolin in T-cells or human colon carcinoma cells also at least 100-150mg are required.
Only thaw aliquots once for usage
AQ 17-5-95
SOLUTIONS
Extraction buffer:
BHT 1000x:
DTPA 1000x:
BAL 1000x:
PMSF 100x:
PBS 10x:
50mM HEPES pH 8, 10% ethyleneglycol, 0.2% Triton X100, 1x

BHT/DTPA
butylated hydroxytoluene (Sigma B-1378) 5mg/ml in ethanol, stored
at 4C
Diethylenetriamine-pentaacetic acid (D-1133) 500mM. Adjust pH to
7 with NaOH.
10mg/ml benzamidine, 2mg/ml antipain, 1mg/ml leupeptin, stored at
-20C
100mM in ethanol, stored at 4C
2g KH2PO4, 4g KCl, 160g NaCl, 28.4g NaH2PO4.2H2O per liter.
After dilution pH should be 7.2 to 7.4.
58
T-CELL PREPARATION
Isolate spleen from normal Balb C mouse (contains about 108 B and T cells)
Homogenize in a potter (= all glass homogeniser with rounded glass pestle) in
DMEM medium/5% FCS, wash cells by resuspending in same medium after centrifugation
(clinical centrifuge 20C,1500 rpm); resuspend cell pellet in cold RBRC buffer for 5 min.
and lyse red blood cells at 4C.
Resuspend cells in 10ml DMEM/10% FCS, let cells sediment for 10 min. at 20C
and spin again at 1500rpm.
Take supernatant and wash 1x in DMEM/10% FCS. Resuspend in 10ml
DMEM/10% FCS and distribute between 4x25 ml flasks (2.5ml per flask). To each flask
add 2.5ml DMEM/10% FCS containing 4ug/ml ConA (Pharmacia # NO17-0450-01)
Incubate for 3 days at 37C in incubator with flasks in upright position.
After 3 days spin cells (mainly T-cells) down in clinical centrifuge, 5 min., 20C at
2000 rpm. Wash 1x by resuspending in PBS at 20C and centrifuging again as above.
Resuspend pellet in 5ml PBS. Underlay PBS with Ficol (Sigma #NR 10771) at RT. Spin 10
min., 2000 rpm, 20C without brakes. Live T-cells are at interphase, dead T cells at the
bottom of the tube. Collect interphase, wash 2x with PBS to remove Ficol. Yield, about 10 7
cells.
SOLUTIONS
RBRC
PBS
: 13mM NaHCO3, 156 mM NH4Cl, 127 mM EDTA

: 1x
AQ 4-5-95
59
T-CELL LABELING WITH 35S METHIONINE/CYSTEINE

In the case of T-cells, after growing in DMEM complete medium containing ConA
the live cells are separated from dead cells by spining them on a Ficol cushion (See T-cell
preparation). The live cells accumulate at the interphase (about 10 7 cells after starting from
a single spleen)
Collect interphase and wash 3x with methionine/cysteine-free medium (GibcoBRL); spin each time at 2000rpm, 20C, clinical centrifuge
Preincubate in the methionine/cysteine-free medium containing 5% dialysed FCS
(fetal calf serum) for 1 hour at 37C
Spin in clinical centrifuge at 2000 rpm, 20C for 5 min., take cell pellet up in 2-3ml
methionine/ cysteine-free medium containing 5% dialysed FCS, to which 400uCi of 35S
Meth/Cys (NEN or Amersham) are added. Incubate 4 hours at 37C in B-lab incubator
Spin cells down in clinical centrifuge as above and wash 1x with normal medium
containing methionine/cysteine. To do a subsequent immunoprecipitation, lyse cells in 1ml
of Crumptons Lysis buffer containing 10mM Iodoacetamide and 50 mM PMSF
SOLUTIONS
Crumptons Lysis Buffer:
0.5% NP-40, 0.5% Deoxycholate, 50mM NaCl,

25mM Tris-HCl pH= 8.2
Dialysed FCS:
Normal FCS, dialysed 48hrs against PBS 1x, with 2

changes of PBS
AQ 11-5-95
60
TISSUE ISOLATION
Rats (n= .......) were sacrificed by............................................................... and stored at 4 C
until organs were removed, which occured within 4 hours of death. The following organs
were obtained: brain, lung, heart, liver, testes, ovary, kidney. Spleen were not available
(removed by Tschopps group). The organs liver, kidney, heart and lung were cut into slices
to fit into the screw-cap tubes. This was done on ice. Individual brains or testes, or 4
ovaries fit into a tube. In the case of brain also large falcon tubes containing 5 brains (L)
were put aside for PKC preparations. All samples were immediately flash frozen in liquid
nitrogen once in the tubes and subsequently stored long-term at - 70 C. Samples are only
thawed once for extraction!
Date of Tissue Preparation:
Tissue
Nr. of Tubes
Usage
Brain
Lung
Heart
Liver
Kidneys
Testes
Ovaries
Comments:
61
TISSUE EXTRACTION
To make extracts, thaw individual tubes only once. Place contents in
homogenisation buffer (H), which is the same one that is used to extract PKC from rat
brain for PKC preparations except that TX-100 is added to a final concentration of 0.1%
after homogenisation.
Buffer H: 20 mM Tris-HCl pH 7.5, 0.25 M sucrose, 10 mM EGTA, 1 mM EDTA, 10 mM
beta mercaptoethanol or DTT, 1 mM PMSF and 1x BAL cocktail.
Weigh organ pieces while frozen, cut into small pieces and mix 1:5 with ice cold
homogenisation buffer. Homogenize using a Wheaton or Dounce homogenizer with teflon
pestel connected to stirrer. For brain it is sufficient to go up and down about 8x; other
tissues may require more. Perhaps, homogenize further by sonication: 3 x 20 sec. at energy
level 3-4. Finally, add TX-100 to a final concentration of 0.1% and stir for 1 hr at 4 C.
Spin at least for 30 min in Eppendorf centrifuge at 4 C. Separate supernatant and pellet.
Resuspend pellet in the same volume of buffer and homogenize again briefly to get more or
less into solution. Make aliquots of both sups and pellets from each tissue. Flash freeze in
liquid nitrogen, store at -70 C. Thaw aliquots only once. Do protein determination. For
Western Blot analysis either using anti-PKC peptide antibodies (antibody characterization)
or GST-PKC fusion proteins (PKC binding proteins) in overlay assays run 30 - 50g of
protein per lane. Do CHCl3/CH3OH ppt to remove TX-100 detergent for gel samples. Run
12.5 % SDS-PAGE to analyze samples.
Date of Extract Preparation:
Tissue weight
supernatant pellet
(g)
(vol./conc)
Brain
Usage
(vol./conc)
s
p
s
p
s
p
s
p
s
p
Lung
Heart
Liver
Kidneys
Testes
s
p
s
p
Ovaries
62
PREPARATION OF BAL 1000X STOCK

Leupeptin stock: To 5 mg add 2 ml dd water (conc. 2.5 mg/ml)
Antipain stock: To 10 mg add 1 ml dd water (conc. 10 mg/ml)
Benzamidine: To 100 mg add 1 ml dd water (conc. 100 mg/ml)
The leupeptin and antipain stock can be stored frozen at minus 20C. Do not freeze-thaw
more than about 2x.
mix
220 ul benzamidine
880 ul leupeptin stock
440 ul antipain stock
760 ul dd water
This mix is equivalent to BAL 1000x. Aliquot the mix into 40 tubes with 50-55 ul per tube.
Store frozen at -20C. Thaw these aliquots only once.
63
16-BAC/SDS-PAGE: A TWO-DIMENSIONAL GEL ELECTROPHORESIS

Reference: Anal. Bioch. 240, 126-133 (1996)
The day before runing the gel prepare the Separating gel: (10ml, 7.5%)
1.8g
Urea
2.5ml
30%polyacrylamide/8% bisacrylamide
2.5ml
300mM potasium phosphate buffer pH 2.1
2.5ml
distilled water
350l
1.7% N,N-methylene bisacrylamide
500 l
80mM Ascorbic acid (prepare fresh:14mg/ml in water)
16l
5mM ferrous sulfate (prepare fresh:1.4mg/ml in water)
100l
250mM 16-BAC (0.99g/10 ml in water, warm to dissolve)
Mix well and add 400l of diluted hydrogen peroxide (1/1200 in water from 30%
stock)
Leave polymerizing for 30 min at RT. Overlay then with 75mM potasium phosphate buffer
pH 2.1 (dilute 4x the buffer 300mM)
Store overnight at 4C
Prepare 500 ml 10x BAC-running buffer
4.95g BAC
56.3g Glycine
28.8ml phosphoric acid (85%)
Stacking buffer (4%, 5ml)
0.5g
Urea
665l
30%polyacrylamide/8% bisacrylamide
1.25ml
500mM potasium phosphate buffer pH 4.1
1.5ml
distilled water
690l
1.7% N,N-methylene bisacrylamide
250 l
80mM Ascorbic acid (prepare fresh:14mg/ml in water)
4.25l
5mM ferrous sulfate (prepare fresh:1.4mg/ml in water)
35l 250mM 16-BAC (0.99g/10 ml in water, warm to dissolve)
Mix well and add 250l of diluted hydrogen peroxide (1/750 in water from 30%
stock)
Leave polymerizing for 30 min at RT. Wash wells with running buffer1x
Get it ready to be load with samples. Wells that do not contain samples need to be filled
with sample buffer to avoid distortion of the gel
Sample buffer 1x (prepare fresh and 1x if possible since it is difficult to solubilize)
0.225g
Urea
50mg
16-BAC
50l
glycerol
200l
distilled water
Dissolve this first (warm up, max. 60C), then add
25l
1.5M DTT
5l
5% pyronin
Bring to 1ml with water and use as soon as possible
SAMPLES
Cell Extracts
Cells (107) are solubilized in 400 l of SB1x (Add 1mM Vanadate and 5mM EDTA)
Incubate at 60C for 5min
Spin and load 45l per lane
Cell Membranes
Membranes (from 2x107 cells) prepared by cavitation are in a pellet.
64
Thaw pellet and resuspend in 400 l BAC sample buffer. Add 1mM Vanadate and 5mM
EDTA
Incubate at 60C for 5min
Immunoprecipitates
Resuspend beads in 100 l BAC sample buffer. Add 1mM Vanadate and 5mM EDTA
Incubate at 60C for 10min. Vortex in between
Run samples to cathode (opposite to SDS-PAGE)
Current 15mAmps/gel until dye front enters separating gel then at 30mAmps/gel until the
dye front has completely run out of the gel (1.5h)
Prepare fixative solution:
3.5 : 1 : 5.5 Isopropanol: Acetic acid: water
Coommassie blue R250 is prepared in fixing solution at 0.15%
-Take the gel off the plates carefully with the stacking
-Put it in fixative solution for at least 1h changing the solution 4x to remove detergent
-Stain the gel for 15min with coomassie blue in fixative
-Then destain in fixative
NOTE: the gel can stay at 4C than any of the last 3 steps
Buffers
0.3M H3PO4 (85%): 3.46ml in final 100ml (water)
0.5M KH2PO4:
0.5M K2HPO4:
0.3M KH2PO4: 60ml of 0.5M KH2PO4 in final100ml (water)
Buffer 0.3 M Potassium Phosphate: take 50 ml of 0.3M KH2PO4 and titrate to pH 2.1 with
0.3M of H3PO4
Normal SDS-PAGE
- Carefully cut 2 strips with a glass plate
- Transfer strips to reequilibration buffer (100mM Tris-HCl pH6.8) incubate 10 min and
change buffer 3x.
-Prepare two 10% gels using the comb with one small tooth and one big tooth.
-Mount in chamber and filled with normal Laemli buffer. Leave big tooth dry and add a bit
of SB1x+10mM DTT
-Put strips of preequilibrated gel in 2 big pieces of parafilm and add 3x SB+DTT. Let the
strip slide into the big tooth with the stacking toward the little tooth where the MW
standard will be added. Push the gel down gently with the same parafilm or a tiny spatula.
There should be no bubles, no space between the 2 pieces of gel
- Overlay the gel piece with 3x SB+DTT and incubate for 5 min
-Add MW standard to the small tooth
- Run gel at 5mAmp/gel
- There will be 2 dye fronts. When the second (slowest) enters the separating gel the current
can be increased upto 20mAmp/gel.
- The gel is ready when the second dye front reaches the bottom.
Both gels are then tranfered to PVDF membranes at 80V for 2 hrs, 4C
65
Immunoblotting
Gel 1
2% BSA inWB to block for 1h. Wash the blot with washing buffer and probe
with Streptavidin-HRP (1/20000 in WB). Incubate for 30 min. Wash 5x10min and develop
with ECL
Gel 2
2%gelatin in WB to block for 1h. Probe with 4G10 (1/2000 in WB). Wash
5x10min and then add the anti-mouse-HRP (1/2000 in WB). Incubate for 1h. Wash
5x10min and develop with ECL
66
BIOTIN (NHS) LABELLING OF PROTEINS

1) Biotin at -20C. Let it reach room temperature before opening
2) Protein has to be in 50mM bicarbonate buffer pH 8.5. Either adjust by adding a more
concentrated solution or dialyze the protein (Centricon)
Ideal concentration of the protein = 2mg/ml
3) Prepare Biotin (ImmunoPure NHS-Biotin, Pierce N21217). 1mg of Biotin is
solubilized in water 1 ml. Immediately add it to the protein. Mix by inversion.
Add 74l/ml of protein solution
4) Incubate for 30 min at room temperature or for 2 hrs at 4C
From now on keep everything at 4C.
5) To stop reaction dilute the solution with 10mM Tris pH 7.4/0.15N NaCl
6) Then dialize the protein with PBS (Centricon) to eliminate the biotin
7) Adjust final volume, add azide to keep at 4C
67
BRADFORD PROTEIN ASSAY

Standard curve is done starting with a BSA solution at 1.39 mg/ml
1- Dilute BSA stock 7x (50 l BSA + 300 l distilled water) to have a solution at 0.2 mg/ml
2- Prepare 4 tubes with 0 protein (for blanks) and the rest in duplicates as follows:
Tube N
1- 4
5- 6
7- 8
9- 10
11-12
13-14
15-16
17-18
19-20
g BSA/ml
0
1
2
3
4
5
7.5
10
12.5
l BSA (0.2mg/ml)
l dd water
0
2
4
6
8
10
15
20
25
3- Prepare dilutions of the samples in a final volume of 400l

Tube N
l sample
l dd water dilution
A 620nm
1
2
3
4
5
6
7
8
400
398
396
394
392
390
385
380
375
g/ml
4- Vortex all tubes

5- Add 100 l of Bradford reagent per tube (BioRad)
6- Vortex again all tubes and leave at room temperature for 15 min
7- Distribute samples on a 96 wells plastic plate of low protein binding (180l per well).
All row N1 is for blanks.
8- Read in the ELISA reader, Dynatech MR5000
Use test N 29 called 413. Edit it first to make sure that the blanks are in A1, B1,C1, D1,
E1, F1, G1, and H1. Filter is 620nm.
68
STOCK SOLUTIONS FOR RIPA BUFFER

Concentration
amount/vol.
Tris-OH, pH 7.4
1M
6 g/ 50 ml
NaCl
1.5 M
4.38 g/ 50 ml
Sodium Deoxicholate
10%
5 g/ 50 ml
Na3VO4
50 mM
0.46 g/ 50 ml
EDTA
1M
18.6 g/ 50 ml
Nonidet P-40
10%
5 ml/ 50 ml
SDS
10%
5 g/ 50 ml
50 ml of 2x RIPA buffer
10 ml 10% NP-40
10 ml 10% DOC
1 ml 10% SDS
5 ml 1M Tris-OH
10 ml 1.5 M NaCl
14 ml water
10 ml of RIPA buffer (prepare just before use)
20 Fl 10 mg/ml Leupeptin
10 Fl 1M PMSF
100 Fl Aprotinin
100 Fl 50 mM Na3VO4
20 Fl 1 M EDTA
69
BUFFER TRITON TO WASH IMMUNOPRECIPITATES

MW
Concentration
amount/vol.
Tris-OH
121.1 g
50 mM
0.606 g/100 ml
NaCl
58.44 g
0.6 M
3.5 g/ 100 ml
0.5 %
0.5 ml/ 100 ml
Triton X-100
PBS (PHOSPHATE BUFFER SALINE)

amount/500 ml
NaCl
58.44 g
40 g
Na2HPO4
5.75g
KH2PO4
1g
KCl
1g
CM (CULTURE MEDIA)
amount/ 500 ml
HEPES, sodium salt 20 mM
HEPES, acid
2.493 g
20 mM
2.383 g
99.37 mM
2.904 g
KCl
4.78 mM
0.178 g
CaCl2.2H2O
(anh)
1.71 mM
KH2PO4
1.19 mM
MgSO4.7H2O
(anh)
1.19 mM
Penicillin
6 U / ml
17.9 mg
Streptomycin
60 Fg/ml
30 mg
Sodium Lactate
25 mM
2.2 ml
Sodium pyruvate
1 mM
58 mg
NaCl
58.44 g
D-Glucose
0.126 / 0.095 g
0.081 g
0.147 / 0.072 g
0.5 g
Check ph 7.4, add a tip of phenol red as ph indicator. Make aliquots of 40 ml and freeze (70
70oC). When thawing, add 0.8 g BSA, 83 mg NaHCO 3, filter and equilibrate o.n in 5%
CO2/ air incubator. Frozen aliquots are good for 6 months. Complete media is good for 2
days.
OTHER BUFFERS
MnCl2.4H2O
Na3VO4
MW
Concentration amount/volume
197.9g
0.5 M
98.95 mg/ml
0.2M
39.58 mg/ml
10 mM
1.84 mg/ml
50mM
9.2 mg/ml
183.9g
ATP
551.1g
0.1 M
55.1 mg/ml
CaCl2.2H2O
147.0g
0.2 M
29.4 mg/ml
MgCl2.6H2O
203.3g
0.2 M
40.66 mg/ml
0.5M
101.65 mg/ml
71
HEPES BUFFER
acid
238.3g
20 mM
0.47 g/100 ml
salt
260.3g
20 mM
0.52 g/100 ml
mix 60% acid + 40% salt for a 20 mM HEPES buffer pH 7.2-7.4.

TN BUFFER
Tris-OH
NaCl
20 mM
58.44g
150 mM
1.21 g
3.8 g
Adjust pH at 7.4 and complete volume to 500 ml.

CHAPS EXTRACTION BUFFER
For preparation of 50 ml
amount
Chaps
20 mM
0.615g
NaCl
50 mM
0.146g
Tris-OH
10 mM
60 mg
EGTA (0.2 M)
2 mM
0.5 ml
PMSF (0.1 M in EtOH)
1 mM
0.5 ml
Benzamidine (0.1M)
1 mM
0.5 ml
0.5 mM
0.5 ml
Na3VO4 (50 mM)
72
PREPARATION OF GST-PBD BEADS (FOR CDC42 AND RAC ASSAYS)

1) Growth of bacteria
prepare ON culture of pGEX GST-PBD (20 mL LB with 50-100 ug/mL
ampicillin), incubate ON to 37oC
dilute ON culture 1:10 into a final volume of 200 mL LB with 50-100 ug/mL ampicillin
grow culture for a further 1-2 hours to 37oC (OD a 600 nm should be about 0.6-0.8)
induce protein expression by adding 0.3 mM IPTG for 3 hours to 37oC
2) Purification of GST-PBD on Glutathione Sepharose 4B
pellet bacteria by centrifugation a 4000 rpm for 10 minutes to 4oC
resuspend pellet in lysis buffer:
20 mM HEPES, pH 7.5
120 mM NaCl
10% glycerol
2 mM EDTA
1 mM PMSF
10 ug/mL each of aprotinin and leupeptin
sonicate suspension 2X, 15 seconds each
spin a 15000 rpm to 4oC for 30 minutes
to supernatant add NP-40 to 0.5%
add 600 uL of a 50% slurry of glutathione-Sepharose 4B beads
incubate 1 hour to 4oC with rotation
wash beads 5X in lysis buffer with 0.5% NP-40
wash beads 3X in lysis buffer with no NP-40
store to 4oC for up to 1 week as a 50% slurry in lysis buffer with no NP-40
3) analysis of GST-PBD product
measure the protein concentration of the beads (should be 2-6 mg/mL)
run fusion protein on SDS-PAGE gel and stain with commassie (fusion protein should
run around 33 kDa)
73
RAC1 AND CDC42 ACTIVITY ASSAYS
use enough cells to yield 800-1000 ug total protein (1 10 cm2 plate for most cell types)
lyse cells in 300 uL of Buffer B
50 mM Tris, pH 7.6
150 mM NaCl
1% Triton X-100
0.5 mM MgCl2
+ inhibitors (PMSF, leupeptin, aprotinin)
sonicate lysates for 10 second only if using suspended cells
spin for 4 minutes to 14000 rpm to 4oC
measure protein concentration (use equal concentration and volumes)
add 30 ug GST-PBD*
incubate to 4oC with rotation for 30 minutes
wash beads 4X in Buffer B
carefully aspirate off supernatant
suspend beads in 30-90 uL of SDS-PAGE sample buffer, boil 5 minutes
run sample on 15% SDS-PAGE gel, transfer to PVDF**
block 1 hour to ON in TBS-T + 5% milk
blot GST-PBD pull-downs with mAb anti-Rac (1:1000) or mAb anti-Cdc42 (1:250).
All antibodies are from Transduction Laboratories.
*Rac1 and Cdc42 activity can be assessed from the same GST-PBD pull-down. To do this
use 1/3 of the sample for the Rac1 blot and 2/3 of sample for Cdc42 blot.
** PVDF must be prepared before transferring. This is done by (1) washing with methanol
15 sec., (2) rehydrating with water 1 min., (3) soaking in transfer buffer for 2 min. or longer
74
STIMULATION OF ....... CELLS (IP AND ENDOF TREATMENT)

1) Take 40 x 106 of ........ cells. Spin cell suspension 5min at 2000 rpm, RT. Discard
supernatant.
2) Wash twice with PBS. Resuspend at 1 x 107 cells/ml in PBS/2.5%FCS
3) Place 1x107 cells (1ml) per eppendorf tube and add the following:
Tube#
Add
1__________________________________________
2__________________________________________
3__________________________________________
4__________________________________________
4) Incubate for 2 min at 4C
5) Spin max speed few seconds ( ). Discard supernatant.
6) Add second antibody warm.
Tube#
Add
1__________________________________________
2__________________________________________
3__________________________________________
4__________________________________________
7) Incubate for 2 min at 37C. Spin max speed few seconds ( ). Discard supernatant.
8) Add 800l of cold PBS/5mM EDTA/1mM Vn quickly mix and spin at max speed for
15 sec.
9) Resuspend samples 1 and 2 in 500 l of Chaps buffer and samples 3 and 4 in 500 l of
RIPA buffer (2ml 1x RIPA + 40 l Vanadate 50mM, 20 l 0.5M EDTA, 2l
BAL:100g/ml benzamidine/ 10g/ml Antipain/2.5g/ml Leupeptin)
From now on keep cells at 4C.
10) Sonicate. Leave at 4C for 15-60 min
11) Spin 5min max speed and eliminate the pellet.
IMMUNOPRECIPITATION
1) To each of extract add Protein A-agarose (50-100l beads)
2) Incubate for 1 hr at 4C. Rotating
3) Wash the beads with lysis buffer 3x and once with PBS
EndoF TREATMENT
1) Add 50 l of 1% SDS in 50mM Tris pH 7.4 containing Vanadate (1mM) and EDTA
(5mM)
2) Boil 3 min, quickly spin, let cool down
3) Add 450l 0.5% NP40 in phosphate buffer pH 7.2 containing NaN3, Vanadate and
EDTA. Mix well
4) Add 75 U of Nglycosidase F (0.5 l of a preparation of 125 U/l) only to one tube
5) Incubate at 37C ....hr
6) Quickly spin. Separate and save supernatant and pellet
7) Supernatant : chloroform-Methanol precipitate it all. 500l methanol+100l chloroform
+ 500l sample. Dry pellet and resuspend in 60l of SB non-reducing agent.
Vortex.Spin. Separate sample in two and add reducing agent to one of them
Pellet: Boil beads in 60l of SB non-reducing agent. Vortex.Spin. Separate sample in two
and add reducing agent to one of them
8) Run samples on a gel
75
CHOLESTEROL EXPERIMENT: STIMULATION OF EL-4 CELLS AFTER

CHOLESTEROL DEPLETION (ANTI-PY BLOT)
-Assay buffer: RPMI + 2% BSA 99%fatty acid free
-Prepare a solution of 100mg/ml of -methyl-cyclodextrin (-CD) in assay buffer.
To solubilize warm it up to 60C for 10min. Let cool down at room temperature before use.
1. Take 20 x 106 of EL-4 cells. Spin the cells (2000 rpm, 5 min, RT) and wash them twice
with RPMI (no protein). Resuspend at 107 cells/ml
2. Place 3x106 cells (300l) per eppendorf tube, spin and withdraw supernatant.
Resuspend as follows:
Tube#
Add
-CD(100mg/ml)
Assay buffer(l)
1__________________________________________
2__________________________________________
3__________________________________________
4__________________________________________
5__________________________________________
6__________________________________________
3. Shake 30min RT. Spin 2000rpm for 5min. Wash cells with assay buffer (2x, 500 l)
4. Resuspend in 320l of assay buffer and separate each sample in 3 aliquots of 100l.
Label 1-6A, 1-6B, 1-6C
5. Stimulate cells as follows
Tube#
Add
A
B
C
1__________________________________________
2__________________________________________
3__________________________________________
4__________________________________________
5__________________________________________
6__________________________________________
6. Incubate for 2 min at 4C
7. Spin max speed few seconds ( ). Discard supernatant.
8. Add 100l of warm second antibody at 100g/ml.
9. Incubate for 2 min at 37C.
10. Add 800l of cold PBS/5mM EDTA/1mM Vn quickly mix and spin at max speed for
15 sec.
11. Resuspend in 100l of RIPA buffer(2ml 1x RIPA + 40 l Vanadate 50mM, 20 l 0.5M
EDTA, 2l BAL:100g/ml benzamidine/ 10g/ml Antipain/2.5g/ml Leupeptin)
12. Sonicate. Leave at 4C for 15-60 min
13. Spin 5min max speed and eliminate the pellet.
14. Supernatant is then precipitated with Chloroform-methanol technique: 100 l, 1 x106 +
400l methanol+100l chloroform+ 300l water.
15. Dry samples, resuspend them in 60 l of sample buffer + reducing agent +Vn. Load on
a gel (28l,0.5x106 cells/lane).
76
Chromatographic parameters
Sample:_________________mg/ml
Protein concentracion_______mg/ml
Total protein loaded________g
Injected volume___________l
Column_________________
Dimensions______________
Particle diameter__________
Column Volume__________
Void Volume_____________
Eluent__________________
pH_____________________
Gradient________________
_______________________
_______________________
_______________________
Flow rate_______________
Detector range___________
Recorder speed__________
Temperature____________
Fraction volume__________
Instrumentation
Pump: 2150 HPLC
Detector: 2158 Uvicord SD
Recorder: 2210 Recorder 2-channel
Fraction collector:2212 Helirac
Rac#_______
Mode_______
Fraction: number______
size_________
77
GEL SCANNING
1) Open Applescan application(in Hard disk). Scanner has to be ON
2) From previsualization window choose:
Resolution=150dpi
Niveaux de gris
Reglages:selection des gris (normal)
3) Previsualiser (image appears in Applescan window)
4)Selectioner (select part of image you want to scan, the smaller the better, otherwise it uses
to much memory)
5) Numeriser (a new window from the file menu has to be open in case this option is
inactive)
6)Go to the window sans titre and enregistrer sous. Copy on the hard disk or in a floppy.
Choose TIFF format.
PROCESSING IMAGE
1) Open NIH Image program (load macros: Gel plotting macros)
2) Import image in TIFF
Custom (varies depending size of image)
Offset= 102 (also varies)
8-bit
calibrate
When using some versions of this program it says file error 39? The image is there and it
can be used but it shows in duplicate.
3) select area to measure (tool )
4) spec/macro/setup to plot gel
5) Number of lanes 1
6) plot background lane
7) Mark areas with the line tool ( )
8) Measure with the wand tool ( )
78
IODOBEAD PROTEIN IODINATION

Antibody or protein concentration should be between 0.8-1.2 mg/ml.
protein:125I to use is 1 g/ml: 10 Ci.
The ratio
Poure and equilibrate a Sephadex G-50 Fine (~4 ml) in a small plastic column using PBS0.4% PVP as a buffer. Keep it cold. (Protein should have a Mr>30000 Da. Otherwise, use
appropriate Sephadex which exclude your protein)
Prepare a 10 mg/ml L-Tyrosine saturated solution.
Iodobeads: 1-2 beads per
g of protein. Rinse the beads in buffer phosphate, pH 7
(best pH for iodination is 6.5 ) and remove excess of liquid with a filter paper. Place beads
in an eppendorf tube.
ALL FOLLOWING STEPS SHOULD BE CARRIED OUT IN A WELL VENTILATED
HOOD.
Assuming that the protein is in buffer phosphate, pH 7:
1) Add, at the same time, 50 l of the phosphate buffer (example 0.1 M sodium phosphate)
and 500 Ci of the Na125I into the iodobead containing tube.
2) Incubate for 5 min tapping the tube from time to time.
3) Add 50 l of the protein solution and incubate for 5-15 min at room temperature (longer
incubation only leads to oxidation of the protein).
4) Transfer the mix immediately to an eppendorf tube containing 100 l of saturated
tyrosine.
5) Shake the mix and load onto the gel filtration column. Use cold PBS as column buffer.
6) Recover fractions of 500 l and the labelled protein should be in the second fraction.
7) Discard column and all radioactive waste in appropriate containers.
Protein labelled____________________________________________________________
Fraction #
cpm/l
79
IODOGEN PROTEIN IODINATION

Antibody or protein concentration should be between 0.8-1.2 mg/ml.
protein:125I to use is 1 g/ml: 10 Ci.
The ratio
Pure and equilibrate a Sephadex G-50 Fine (~4 ml) in a small plastic column using PBS0.4% PVP as a buffer. Keep it cold. (Protein should have a Mr>30000 Da. Otherwise, use
appropriate Sephadex which exclude your protein)
Prepare a 10 mg/ml L-Tyrosine saturated solution.
Iodogen: 1 mg/ml Iodogen (1,3,4,6-tetrachloro 3 ,6 -diphenylglicouril) in chloroform.
Make aliquots of 60 l in eppendorf tubes and leave inside the hood o.n. to dry. Store in
desiccator at room temperature. Stable for several years.
ALL FOLLOWING STEPS SHOULD BE CARRIED OUT IN A WELL
VENTILATED HOOD.
Assuming that the protein is in buffer phosphate, pH 7:
1) Add, at the same time, 50 l of the protein solution and 500 Ci of the Na125I in a
iodogen-coated tube.
2) Incubate for 2 min tapping the tube constantly (longer incubation only leads to oxidation
of the protein).
3) Transfer the mix immediately to an eppendorf tube containing 50 l of saturated
tyrosine.
4) Shake the mix and load onto the gel filtration column. Use cold PBS as column buffer.
5) Recover fractions of 500 l and the labelled protein should be in the second fraction.
6) Discard column and all radioactive waste in appropriate containers.
Protein labelled___________________________________________________________
Fraction #
cpm/l
80
KINASE BUFFER
DTT (0.5M,100x)
Na3VO4 (50mM,250x)
cAMP-Kinase inhibitor (100M,100x)
MnCl2 (0.2M,125x)
CaCl2 (0.2M,100x)
Leupeptin (10mg/ml,100x)
Aprotinin (500KIU,100x)
50 l
20 l
50 l
125 l
50 l
50 l
50 l
Final concentration
5 mM
200 M
1 M
5 mM
2 mM
100 g/ml
5 KIU
Complete volume to 5 ml with 20mM Hepes Buffer pH7.2
81
LABELLING OF ASTROCYTES MEMBRANES WITH BIOTIN (NHS)

1. Biotin at -20C. Let it reach room temperature before opening
2. Use astrocytes membrane resuspended in...... ml of PBS (1ml in a 15ml falcon tube).
Membranes are from ............... number of cells
3. Prepare Biotin (ImmunoPure NHS-Biotin, Pierce N21217).
1mg of Biotin is
solubilized in water (0.9 ml) and then 10x PBS (100l) are added. Immediately add it
to the membranes. Mix by inversion.
Cell concentration =2.5 x107 /ml
Biotin concentration= 0.5mg/ml
4. Incubate for 30 min at room temperature
5. Add 4-5 volumes of buffer 10mM Tris pH 8.0/0.15N NaCl
6. Mix and spin suspension at 30000 rpm, 4C for 30min. Discard supernatant.
7. Resuspend in ...... ml of ............... buffer with Vanadate and BAL.
Save it at -70C after snap freezing it or use immediately.
82
MEASURING ESTERASE ACTIVITY OF ACROSIN

Note: Acrosin sticks strongly to glass. Siliconize quartz cuvettes and all glass material to
use. Acrosin also undergoes autodegradation at neutral pH, it is recommended to keep it a
pH 3.0
References
Can. J. Biochem. 60:8-14 (1982)
Biochem. Biophys. Acta. 16: 570-575 (1955)
Mix directly in quartz cuvette 0.5ml of BAEE 3mM+ 10-100l of acrosin at pH 3.0+
necessary amount of tris buffer (0.1 M Tris-HCl pH 8.0) to complete 3 ml (room
temperature).
Follow change in OD at 253nm for 5-10 min
1 U of acrosin= amount necessary to hydrolyze 1M of BAEE/min.
BAEE= N-alpha-benzoyl-arginine-ethyl-ester (sigma)
83
SPERM MEMBRANE PREPARATION.

MgCl2 Protocol
Recover sperm in PBS, 370C, washed once (600xg, 10 min) and count.
Resuspend in CM for 60 min capacitation and wash again with PBS to eliminate excess of
protein.
Resuspend sperm in 50 mM MgCl2 in water, 40C, leave 30 min in corex tubes.
Spin in sorvall SS-34 10min, 3000 rpm, 40C.
Recover supernatant and ultracentrifuge to pellet membranes (Ti-70.1 rotor, 60 000 rpm, 35
min).
Pellet can be solubilized with detergent containing buffer at ~108 cells/ 100 l of buffer to
give ~ mg/ml of protein.
84
MEMBRANE PREPARATION
Cells
EL-4 GB (Thy-1+)
EL-4 -f (Thy-1-)
Astrocytes
Cell pellets were washed twice with PBS/0.5 mM EDTA/ protease inhibitors and
resuspended in 10 ml of same buffer
Cavitation for 30 min (pressure 30psi)
Spin at 2000 rpm for 15 min, 4C, 2 times
Recover supernatant and spin in ultracentrifuge at 30000 rpm (SW 55Ti, code number 2)
for 30 min, 4 C. This corresponds to 100 000 x g. Tubes used Beckmann ultra clear (13 x
51mm). Order N344057
Discard supernatant. Resuspend pellet in PBS/ 0.5 mM EDTA/ protease inhibitors. At
approx 5 x 108 /ml
Homogenize pellets with eppendorf teflon homogenizer and also using a 1ml syringe with a
26G needle
Make aliquots of 100l and snap freeze in liquid nitrogen. Store at -70C
Save an aliquot of 50 l. Solubilize it with 50 l of PBS/EDTA/BAL+ 0.2% TX-100 +
0.2N NaCl (1ml PBS/EDTA/BAL + 20 l 10% TX-100 + 40 l 5N NaCl)
Measure protein content of preparation by Bradford method.
85
PREPARATION OF GST-RBD (FOR RHOA ASSAYS)

1) Growth of bacteria
prepare ON culture of pGEX GST-RBD (10-40 mL LB with 100 ug/mL amp)
dilute ON culture 1:50 into a final volume of 400 mL LB with 100 ug/mL amp
grow culture for ~1 hours to 37oC (OD600 nm should be roughly 0.2)
induce with 0.3 mM IPTG ~16 h to RT (OD600 nm should be roughly 1.8)
2) Purification of GST-RBD on Glutathione Sepharose 4B
pellet bacteria by centrifugation to 4000 rpm for 10 minutes to 4oC
resuspend pellet in 10 mL lysis buffer:
50 mM Tris, pH 7.4
50 mM NaCl
5 mM MgCl2
1 mM DTT
1 mM PMSF
10 ug/mL each of aprotinin and leupeptin
1 % Triton X-100
split suspension in half (2 X 5 mL) and sonicate 6X, 20 seconds each
spin to 12,000 rpm to 4oC for 10 minutes
rotate supernatant for 1 hour with 1 mL of a 50% slurry of Glutathione Sepharose
4B (wash beads in lysis buffer before adding to supernatant)
wash beads 3X in lysis buffer
wash beads 3X in lysis buffer with no Triton X-100
store to 4oC for up to 1 week or in 50% glycerol to -70oC
3) analysis of GST-RBD product
measure the protein concentration of the beads (should be 3-6 mg/mL)
run fusion protein on SDS-PAGE gel and stain with commassie (fusion protein
should run around 33 kDa)
86
RHOA ACTIVITY ASSAYS
use enough cells to yield 800-1000 ug total protein (1 10 cm2 plate for most cell
types)
lyse cells in 300 uL of Buffer A
50 mM Tris, pH 7.6
500 mM NaCl
0.1 % SDS
0.5 % DOC
1% Triton X-100
0.5 mM MgCl2
sonicate lysates for 10 second only if using suspended cells
spin for 4 minutes to 14000 rpm to 4oC
measure protein concentration (use equal concentration and volume)
add 15-30 ug GST-RBD
incubate to 4oC with rotation for 30 minutes
wash beads 4X in Buffer B
50 mM Tris, pH 7.6
150 mM NaCl
1% Triton X-100
0.5 mM MgCl2
carefully aspirate off supernatant
suspend beads in 30 uL of SDS-PAGE sample buffer, boil 5 minutes
run sample on 15% SDS-PAGE gel, transfer to PVDF*
block 1 hour to ON in TBS-T + 5% milk
blot GST-RBD pull-downs with mAb anti-RhoA (1:250) from Transduction
Laboratories.
* PVDF must be prepared before transferring. This is done by (1) washing with methanol
15 sec., (2) rehydrating with water 1 min., (3) soaking in transfer buffer for 2 min. or longer
87
SPERM MEMBRANE PREPARATION

Cauda epididymides of retired breeder mice are recover in TN buffer/ 2mM EGTA/ 100
M Na3VO4 on ice (0.5 ml /mouse). Sperm are maintained at 0-40C and released by
mincing the epididymides with scissors and shaking gently on a rotating platform for 1015 min. Sperm are transferred to 15 ml Corex glass tubes (only for sperm membranes
tubes) and washed by centrifugation (700xg, 10 min, 2x with the same buffer)(Sorval
centrifuge, SS-34 rotor, use 2500-3000 rpm). Count sperm between this two washes.
Resuspend washed sperm at 1-2 x 108 sperm/ml no more than 2.5 ml/ tube.
Demembranate sperm placing the tube in the vortex at #6 for 2 min. Pellet sperm by
centrifugation (1000xg, 15 min, 40C).
Collect supernatant in 13 ml clear plastic
ultracentrifugation tube (cold tube). Resuspend pellet in 1-2 ml of TN/EGTA/Vanadate

buffer and vortex again as before. Recover again the supernatant and pool it with the
previous one in the ultracentrifuge tube. Spin down membranes in a 70.1 Ti rotor (247
000xg=60 000rpm, 35 min 40C). Discard supernatant and resuspend the membranes in
RIPA buffer or with other detergent buffer (1%) at 1 x 109/ml. Make sure you have
protease inhibitors and the sample is always kept cold. Homogenize with teflon pestle with
approx. 10 strokes. Leave the extract in ice for 1 hour. Recover extract in an eppendorf
tube and spin down maximum speed in microfuge (cold) for 5 min. The supernatant
contains solubilized membranes and this can be used immediately or stored at -200C (snap
freeze in liquid nitrogen first). Avoid freeze-thaw of the sample.
88
SURFACE LABELLING (NHS-SS-BIOTIN) OF EL-4 CELLS

Count cells..................
1) Spin cell suspension 5min at 2000 rpm, RT. Discard supernatant.
2) Wash twice with PBS. Resuspend at 2 x 107 cells/ml in PBS (2 ml)
3) Prepare Biotin (ImmunoPure NHS-SS-Biotin, Pierce N21331). 6mg of Biotin are
solubilized in water (1.8 ml) and then 10x PBS (200l) are added. Immediately add it to
cells. Mix by inversion.
5) Separate samples in 4 eppendorf tubes. Cells are washed twice with cold PBS, twice
with 5mM Glycine in PBS, pH 7.4 and once with PBS
6) Follow next page protocol for stimulating the cells
7) To each of extract add Protein A-agarose (50-100l beads)
8) Incubate for 1 hr at 4C. Rotating
9) Wash the beads with lysis buffer 3x and once with PBS
10) Solubilize with non-reducing sample buffer (35l 2xSB and 25 l dH2O)
11) Run on a 12% gel
89
SURFACE LABELLING OF ASTROCYTES (DI-TNC1) WITH BIOTIN (NHS)

2) Remove medium from the plate and gently add PBS. Wash the cells twice with PBS like
that. Then, dettach cells and resuspend them in PBS.
Count cells..................
4) Resuspend at 5 x 107 cells/ml in PBS (1min a 15ml falcon tube)
6) Incubate for 30 min at room temperature
7) Cells are washed twice with 5mM Glycine in PBS, pH 7.4 and twice with PBS.
Resuspend in 1 ml of RIPA buffer with Vanadate and BAL. Sonicate, spin, take the
supernatant and save it at -70C after snap freezing it.
90
SURFACE LABELLING OF EL-4 CELLS WITH BIOTIN (NHS)

Count cells..................
3) Wash twice with PBS. Resuspend at 5 x 107 cells/ml in PBS (1mlin a 15ml falcon tube)
5) Incubate for 30 min at room temperature
6) Separate sample in 4 eppendorf tubes (4x490l. Save the rest to make control nonstimulated: spin and save pellet at -20C). Cells are washed twice with cold PBS, twice
with 5mM Glycine in PBS, pH 7.4 and once with PBS. Resuspend in 1 ml of PBS/2.5%
FCS per tube
Stimulation: prepare before starting the second antibody dilution and warm up at 37C.
Also the CHAPS buffer with BAL (1/1000) and Vanadate (1/50) and keep 4C. Have also
close by at 4C the PBS/Vn/EDTA
1) Take 1-1.25 x107 cells (1ml) per eppendorf tube and let them cool down on ice for 5
min. Once cold, over the lid of the tube, add the following:
Tube#
Add
1__________________________________________
2__________________________________________
3__________________________________________
4__________________________________________
2) Close the lid carefully and mix by inversion all 4 tubes at the same time
4) Spin at max speed 15 sec. Discard supernatant
5) Add 1 ml of second antibody (2.5l goat anti-rat per ml of PBS/FCS) per tube
6) Resuspend the cells as quickly as possible and incubate at 37C for exactly 2min (Take
10 l of cells per tube, pooled, spin, discard supernatant and save these cells at -20C for
control stimulated cells). Spin at max speed 15 sec. Discard supernatant
7) Add 800l of cold PBS/5mM EDTA/1mM Vn quickly mix and spin at max speed for 15
sec.
8) Resuspend in 500l of CHAPS buffer(3ml 1x CHAPS/EGTA + 60 l Vanadate 50mM,
3l BAL:100g/ml benzamidine/ 10g/ml Antipain/2.5g/ml Leupeptin)
91
CHAPS/EGTA buffer: 0.154g CHAPS+ 1.5ml 5N NaCl + 500l 1MTris pH7.4 + 100l
MgCl2 + 2ml 125mM EGTA. Complete to 50 ml with water
9) Sonicate. Leave at 4C for 15-60 min
10) Spin 5min max speed and eliminate the pellet. Pool 2 tubes (800l)
11)Snap-freeze the supernatant (liquid nitrogen) and save at -70C
Prepare the first part (separating) of the BAC-gel and save for the next day at 4C. See
BAC 2-D gel protocol.
Immunoprecipitation
1) Prewashed Protein-A beads three times with PBS 1x
2)Take 800l f thawed extract in their eppendorf tubes and add 100l prot-A beads:
3) Incubate for 1hr at 4C on the rotating wheel.
4) Spin beads 15 sec, max speed
5) Wash the beads with cold CHAPS buffer 3x incubating. Then wash 2x with cold PBS
6) Solubilize proteins from the beads with BAC-sample buffer (100l)
7) Run on a 7.5% BAC-gel (see protocols). Load 45l per lane (4 lanes in total)
92
THY-1 PURIFICATION FROM EL-4GB RIPA EXTRACTS

Equilibration buffer: 1% Sodium Deoxycholate in binding buffer
Binding buffer: 50mM Sodium borate buffer pH 8.2
Washing buffer 1: 50mM Tris-HCl pH 8.0
Washing buffer 2: 50mM Tris-HCl pH 8.0/1% octyl-glucopyranoside
Elution buffer: 50mM Glycine pH 3.0/0.25M NaCl/1% octyl-glucopyranoside
1) Equilibrate the column (2ml of DMP crosslinked anti-Thy-1-Prot A-Agarose, rabbit 287
serum purified. Column was prepared as indicated by Pierce with Ab-orientation Kit)
with 50 ml of Washing buffer 1, Then with 10 ml equilibration buffer.
2) Meanwhile prepare the cell extract. Approx. 6 x 108 cells in a PBS washed pellet are
solubized in 12 ml of equilibration buffer. Solubilize by vortex and sonication and
leave the extract at 4C for 30 min. Spin to remove the nonsolubilized material.
3) Load the extract onto the column and recover the flow through.
4) Wash the matrix with 10 ml of equilibration buffer, then with 50 ml of washing buffer
1 and then with 10 ml of Washing buffer 2.
5) Prepare tubes for elution: Add 100l of 1M Tris pH 8.0 per tube and labelled them.
Elute the column with Elution buffer and collect fractions of 1 ml. Mix the eluate with
the Tris after every tube collection. Measure protein at OD=280nm and pool those
fraction containing the protein
6) Concentrate and dialyze Thy-1 (use sterile PBS with 50% ethylene glycol, PBS/EG)
using Centricon 10 (Amicon). 3hr spin at 6400 rpm (Sorvall, A 8.24 rotor, 5000xg),
then add 2 ml of PBS/ EG, repeat 5x.
The column should be washed immediately 15 ml of 0.1M Glycine pH 2.8/ 0.5N NaCl
buffer (no detergent), then with 50ml of PBS and 10 of PBS containing azide.
Tube #
A 280nm
Tube #
A 280nm
1-------------5-----------2-------------6-----------3-------------7-----------4-------------8-----------Equilibration buffer:
0.5g of deoxycholate in 50ml of binding buffer
Binding Buffer:
Stock solutions: 0.2 M Boric acid (3g/250ml); 0.2M Sodium
Tetraborate
(10g/250ml).
25 ml of the acid and 5 ml of the salt gives 30 ml of 0.2M borate buffer pH 8.2.
Dilute to 120 ml for a 50mM solution
Washing buffer 1:
Stock solution: 1M Tris pH 8.0
12.5ml of stock for 250 ml gives a 50mM Tris pH8.0
Washing buffer 2:
0.1 g of octyl-glucopyranoside in 10 ml washing buffer 1
Elution buffer:
Stock solution: 0.1M Glycine/0.5M NaCl pH2.8-3.0
Dilute 5ml of stock in 10 ml of water and add 0.1 g of octylglucopyranoside.
93
STRIPPING BLOTS
Blot stripping buffer:
FOR 50 ML: -5 ML 10X PBS
-5 ml 20% SDS
-7,5 l 100% -MeOH (final concentration 2,5 mM)
Strip for 15 min. with strip solution at 60 C.
Rinse with PBS 1X 5-10 min.
Stain with Ponceau Rouge (to verify the presence of proteins ).
Rinse again with PBS 1X.
Block the blot for 1 hr. in block solution.
Reprobe with antibodies of intererst
Modified in Chile by Daniela Galleguillos, July 2001
94
NITRITE MEASUREMENT ASSAY

This protocol measures the accumulated nitrite in culture medium. P. Hausel. It is used for
measurement of nitrite in HEK-293 T cells transfected with puroiNOS (plasmid containing
iNOS gen).
Reagents:
1.- For the standard curve: 500 mM NaNO2 (stock 50X) prepared in nanopure water.
Working solution: 30 M NaNO2 in culture medium (usually RPMI).
Prepare 3 ml of each concentration in order to obtain a duplicate of the curve. Mix well.
Standard curve1:
Concentration (in M)
0
3.75
6.25
10
20
30
ml of 30 M NaNO2
--0.375
0.625
1.0
2.0
3.0
ml of Culture medium
3.0
2.625
2.375
2.0
1.0
---
2.- A Reagent: 14.6 mM Sulfanilamide in nanopure water

3.- B Reagent: 1 mM Naftiletilendiamine in nanopure water
Observation: A and B reagents are termolabile and photolabile compounds. Thus, you
should keep at 4C and wrapped in aluminum foil to protect from light.
4.- 1.4 N HCl2
Materials: 5 ml falcon tubes, 100 x 11 mm glass tubes. recycled 15 ml Falcon tubes (these
recycled tubes has to be washed with distilled water many times and finally once with
nanopure water. They have to be dry).
Equipment: UV-visible spectrophotometer available for several hours.
Non-sterile cold OBS + protease inhibitors. 2X loading buffer.
Biological Material: HEK-293 cells.
Cell culture material: 10% FBS DMEM-H glu medium. 10% FBS RPMI medium
1.- Day 1: Transfect cells (revise transfection protocol) and let the cells to express the
protein in RPMI medium.
Day 2: Withdraw culture medium and keep it in 15 ml Falcon tubes.
2.- Centrifuge the medium for 5 minutes at 13000 rpm to pellet cell debris. Collect the
supernatant. You can store frozen at -20C
Levels reached at inducing iNOS with citokine cocktail in HT-29 cells. They do not reach the concentration
of 6 nM. In that case draw the standard curve including the following concentrations: 0, 0.75, 1.25, 2.5, 3.75,
5 and 6.25
2
It is important to make all these reagents (A, B, NaNO2, stock and HCl) in nanopure water beacuse they are
used to measure trace amounts of nitrite.
95
3.- If protein levels are to be analyzed: collect the cells (tripsinization or scraping). Pellet
the cells at 2000 rpm. Wash twice with PBS + inhibitors. Resuspend in 1X loading buffer.
Measure the protein quantity by amidoschwartz method. Run 30 g of each sample in a
12% acrylamide gel.
4.- Mark the cristal tubes, vortex and add 1 ml of each standard and each sample in those
tubes.
5.- Read A: Add 82 l A reactive + 82 l HCl to each tube. Seal the tube with parafilm and
mix by inverting the tube avoiding the production of bubbles. Read IMMEDIATELY at
540 nm.
6.- Value: (B-A). Draw a graphic with this value versus concentration of each standard.
Find the straight equation and calculate the concentrations of the samples.
This protocol has been scalated from ELISA plate measurement which uses lower volumes
of sample being faster at the same time. If an ELISA reader with 540 nm filter is available,
you should use 172 l of each sample and standard. It is basically the same once you have
the the samples and standards ready to be measured:
4.- Place samples and standards in an ELISA plate by duplicate or triplicate.
5.- Read A: For 172 l of sample or standard curve point, add 14 l of A reactive + 14 l
of HCl. Pipette up and down to mix but without producing bubbles. Measure at 540 nm.
Read B: Add 15 lof B reactive. Wait for 10 minutes and measure at 540 nm.
6.- Value: (B-A).
From Laboratory of Emanuela Felley-Bosco
Established in Chile by P. Lisboa Snchez(July 2001)
96
ISOLATION OF MITOCHONDRIA
Per assay use 20x106 cells. Wash 2x in cold PBS centrifuging each time for 5min at
3000rpm (clinical centrifuge). Then lyse cell pellet in 1ml isotonic lysis buffer on ice in the
presence of protease inhibitors (BAL and PMSF). Transfer to cold homogeniser type B and
homogenize 25x on ice avoiding formation of foam. Tranfer to Eppendorf tube and pellet
non-disrupted cells at 1000 rpm for 1min. Transfer supernatent to fresh Eppendorf tube on
ice and centrifuge 10 min at 8000rpm to remove nuclei in pellet. Tranfer supernatant to
ultracentrifuge tube (see the type in materials) and centrifuge 1h at 4oC and 1000000xg to
obtain mitochondrial pellet (and membranes). Transfer supernatant (cytosol) and
chloroform/methanol precipitate. Resuspend in 1x samples buffer wherebyu volume
depends on protein to be analysed: for cytochrome C 20 l (analyse all in one lane), for
other proteins in 100 l (analyse only 20 l). Nuclear and mitochondrial (membrane)
pellets are resuspended in 100l of samples buffer (minimal volume for sonication) and
sonicated.
97
PURIFICATION OF DIGS
Materials and Solutions:
60-70 % confluent Swiss 3T3 cells in plate of 100 mm ( 2*106 cells).
PBS cold
Rubber policeman
Loose-fitting Dounce homogenizer type A for 2 ml.
Centrifuge Sorvall- Rotor H875
Microcentrifuge 13000 rpm to 4C
Polypropylene tubes 5 ml (Nalgene)
Buffer T: 25 mM MES, pH=6,5; 150 mM NaCl; 2mM EDTA; 1% Triton X-100 ,

1xBAL; 1mMNaVO4; 1mM PMSF.
Buffer OG: 50mM OG( Octyl-B-D-glucopyranoside); 20mM Tris, pH=8,0; 0,5

mMNaCl; 2mM EDTA; 2mM EGTA; 1xBAL, 1mMNaVO4, 1mMPMSF.
Pippete pasteur
Ice
Eppendorf
Method:
-
All steps are done at 4C on ice
Swiss 3T3 cells are placed on a bed of ice and the culture medium is eliminated.
Wash cells twice with cold PBS. Eliminate carefully all PBS after the last wash. Add 1
ml of PBS plus inhibitors to plate and detach the cells using the rubber policeman.
Then the cells are harvested by centrifugation for 30 seconds at 6000 rpm in a
microcentrifuge. The cell pellet is resuspended in 200 l of buffer T and incubated for
20 minutes on ice.
Transfer the cell lysate into a loose-fitting Dounce Homogenizer type A previously
cooled . Homogenize extremely carefully with 10 strokes. Homogenate is centrifuged
for 7 minutes at 2000 rpm in a microcentrifuge. The supernatant (SN1) is kept on ice.
The pellet (P1) is resuspended in 60 l buffer T and centrifuged for 7 minutes at 2000
rpm resulting in a pellet (P2) and a supernantant (SN2). SN1 and SN2 are pooled and
centrifuged for 12 minutes at 12000 rpm resulting in a pellet (P3) and a suupernatant
(SN3). The pellet (P2) is resupended in 60 l buffer OG and then centrifugated at
3000 rpm for 7 minutes resulting in a pellet called nuclear fraction and a supernatant
(SN4). The pellet (P3) is resuspended in 200 l buffer OG and pooled with SN4. Both
are incubated for 50 minutes on ice and then centrifuged for 12 minutes at 10000 rpm.
The resulting pellet is designated a cytoeskeletal fraction and the supernatant is called
98
SN6 (DIG fraction). SN3 is centrifuged for 1 hour in an ultracentrifuge at 100000xg.

The supernantant is called membrane & cytosol fraction and the pellet is referred to as
the DIG fraction. The pelleted DIG fraction is resuspended in SN6 which corresponds
to the octylglucoside-solublized part of DIGs.
Based on protocol by Berangere Saucy/Daniel Legler
Established in Chile by Vicky Monardes July 2001
99
Esquema
100
PURIFICATION OF LIGHT MEMBRANE FRACTIONS BY TRITON X-100

EXTRACTION IN SUCROSE GRADIENT
MATERIALS AND SOLUTIONS:
1.- Start with 3 confluent MDCK cell plates (10 cm). For HT29 cells, use two confluent
plates of 10 cm. If you are going to need anpther cell type, you should need approximately
50 x 106 cells or 10 mg of protein.
2.- A glass loose fitting Dounce type B homogenizer.
3.- Ultracentrifuge polypropilene tubes .
4.- Cold sterile PBS
5.- MNE buffer, ph 6.5 (2.5 mM MES; 150 mM NaCl; 2 mM EDTA)
6.- MNEX (extraction buffer): MNE + 1% Triton X-100. This is a viscous detergent.
Prepare a 10% solution taking 1 ml with a cut pipette tip and disolving it in 9 ml of distilled
water. Protect the solution from light.
7.- BAL 1000X
8.- Sucrose solutions: 90%; 35%; 5%. Dissolve in MNE.
These solutions shoukd be solubilized by heating in a water bath or in a microwave. For the
latter, heat until boliling. Keep at 4C. These solution last for approximately 1 month. Be
sure there is no fungal growth.
101
PROTOCOL:
1.- All steps should be carried out at 4C
2.- For adherent cells: Discard the culture medium from plates. Wash twice with cold sterile
PBS. Keep the plates on ice at all time.
For suspension cells: Centrifuge the cells at 1200 rpm during 5 minutes. Discard the
supernatant and wash the pellet twice with cold sterile PBS.
3.- Collect adherent cells by adding a volume of MNEX (extraction buffer) to the plate.
Then, wipe the cells off the plate with a plastic cell-scraper. Transfer the extract to a 15
ml tube. Add 2 l of 1000X BAL and 1000X PMSF per plate.
NOTE: MNEX VOLUME SHOULD BE 2 ML AND MUST BE SPLITTED INTO EVERY PLATE
WITH THE SAME EXPERIMENTAL CONDITION. FOR INSTANCE, YOU NEED 3 MDCK
PLATES OF 10 CM FOR SEPARATION OF LIGHT FRACTIONS (CAVEOLAE). FOR THIS
REASON YOU SHOULD ADD 0.66 ML PER PLATE TO GET A FINAL VOLUME OF 2 ML.
4.-Place the tube on ice for 20 minutes.

NOTE: WHILE THE TUBE IS ON ICE, ADD THE 90% SUCROSE SOLUTION TO THE
CENTRIFUGE TUBE. THE VOLUMEN OF EACH SUCROSE SOLUTION IS DETAILED IN THE
TABLE BELOW:
SUCROSE SOLUTION
90%
Homogenate
35%
5%
Centrifuge/ 12 ml per tube.

2.0 ml
2.0 ml
4.0 ml
4.0 ml
Centrifuge/ 5 ml per tube.

0.75 ml
0.75 ml
1.5 ml
1.5 ml
5.- Transfer the cells (2 ml) into the homogenizer. Place the bottom part pf the homogenizer
on ice and push down the embolo softly until it reaches the bottom. Count up to ten and
pull up the embolo. Avoid taking the head of the embolo out of the liquid to prevent
making air bubbles. Repeat the process 10 times. This is a CRITICAL step and should be
done carefully to obtain a good isolation. Remember to keep everything on ice at all time.
6.- Transfer the homogenate volume indicated in the table onto the 90% sucrose solution.
Vortex gently to obtain a homogenated solution.
7.- Add the 35% sucrose solution volume as indicated in the table with a Pasteur pipette.
Do it slowly to avoid mixing the phases. It is advisable that you add the solution touching
the tube wall to obtain a constant flux which will produce the minimum mix of the phases.
Repeat the same procedure for 5% sucrose solution.
8.- Place the tubes into the centrifuge adapter and calibrate them.
9.- Once the tubes are calibrated, place them in a centrifuge and set the following program:
200000 x g / 20 hours / 4C
10.- After centrifugation is over, take the tubes out of the centrifuge and put them on ice.
102
11.- Separate the different fractions of the gradient starting from the upper part of the tube.
Collect 0.375 ml fractions from 5 ml tubes.
12.- Analize the fractions by western blot, protein distribution and cholesterol.
Cholesterol measurement:
In scintillation vials, add 3 ml of scintillation solution and the add 250 l of
each fraction. Measure the radioactivity in a scintillation counter (molecular biology
laboratory).
Protein distribution:
Mix 10 l of sample and 10 l of 10X sample buffer. Load these 20 l in a
10% or 12% acrilamide gel. Ponceau red staining allows to determine protein distribution.
Immunowesternblotting:
Perform western blot for the presence of caveolin, actin, fyn among others.
Do not forget to load an antibody control. In the caseof caveolin, use an MDCK cell
extract. In the case of PKCs, use a brain extract.
Note: see scheme below
References
1) Lisanti et al. Meth. Enzymol. 1995, 250: 655-668
2) Rodgers and Rose J. Cell Biol. 1996, 135: 1515-1523
103
FRACTIONATION SCHEME TO ISOLATE DETERGENT-INSOLUBLE

MEMBRANE FRACTIONS IN HT-29 CELLS
1
HT-29 cells,
20-30%
confluent
Wash 2X
ice-cold PBS
3
Steps
Labeling of cells with

14C-cholesterol
1 Cells
2 14C-Cholesterol labeling
1% TX-100 in MNE pH
6.5+ protease inhibitors
at 4C
3 washes
4 Lysis
5 Homogenisation
6 Sucrose gradient
7 Centrifugation
Lysis, 20-30
min on ice
8 Fraction collection
6
Sucrose
gradient
Homogenization,
10X loose-fitting
Dounce
7
5%
20 hours
200 000 x g
4C
8
1
6
35%
45%
(Cell lysate
+ 90% sucrose)
Light
fractions
Caveolae-like
membranes
Heavy
fractions
12
based on a protocol developed by Florent Bender

Established in Chile by Vicky Monardes, july
2001
104
DOT BLOTTING WITH CHOLERA TOXIN, GM1 DETECTION IN RAFT

FRACTIONS.
(Protocol from Claudio Hetz, Switzerland)
Homogenization of samples to be analyzed. The choice of method depends on
the protein to be studied. Manual potter and sonication up to 30% power are
between the mostly used in the labs. Once homogenized, add bromophenol blue
(stock 5mg/ml, diluted 400x for use) to visualize the sample on the
membrane. Nitrocellulose membranes should be used instead of PVDF.
Procedure:
1.- Nitrocellulose membranes properly identified and embedded in the transfebuffer (see
solutions below).
Put the embedded membrane on the top of two layers of filter paper. Let the membrane dry
lightly to have well defined spots on it. Use small volumes (2-5ul) of the samples to be
analyzed.
2.- Once finished, transfer the membrane to the blocking solution.
Blocking:1 h RT 3% BSA in PBS (1M).
3.- Incubate the membrane with cholera toxin-horse peroxidase (Sigma, cat: C-4672),
1:10000 for 30 min R. Develop as a normal western blot. It gives a very high signal within
lipid rafts preparations using only 1 2ul of the samples from CNS. In other tissues it has
to be optimized given the high concentration of GM1 in the brain.
105
D: LIPIDS BIOCHEMISTRY
DAG/CERAMIDE DETERMINATION......................................................................107
DAG/CERAMIDE & PHOSPHATE WORKSHEET...................................................109
ISOLATION OF DGK MEMBRANES........................................................................111
LIPID PHOSPHORUS DETERMINATION................................................................112
LIPOSOME ASSOCIATION ASSAY...........................................................................113
INHIBITION OF DYNAMIC PROTEIN PALMITOYLATION IN INTACT CELLS WITH
TUNICAMYCIN...........................................................................................................114
LONG-CHAIN FATTY ACYLATION OF PROTEINS...............................................126
RECRYSTALLIZATION OF -OCTYLGLUCOSIDE...............................................142
106
DAG/CERAMIDE DETERMINATION
MATERIALS
glass screw cap tubes with teflon seal
nitrogen
nitrogen manifold
water bath sonicator (Branson 1200)
hair dryer
Octyl-b-D-glucopyranoside (Calbiochem #494460 (99%); Sigma #O8001 98% also
works)
Ceramide, brain
(Avanti Polar Lipids #860052)
Phosphatidylglycerol, diC18:1 (Avanti Polar Lipids #840475)
E. coli membranes with DGK
Imidazol (Fluka #56748)
ATP, disodium (Sigma #A2383)
32
P-gATP (Amersham #PB10218; 5000 Ci/mol, 10 Ci/ml)
TLC chamber (Sigma #z12,619-5)
MERCK Silica gel 60 TLC plates (Sigma #z29,297-4)
Kodak film and exposure cassettes
organic solvents chloroform, methanol, pyridine and acids HCl, HCOOH (Fluka,
99.5% by GC)
PROCEDURE
1. Isolation of cellular lipds (Bligh & Dyer, 1959, Can. J. Physiol. 37: 911-917).
- Use roughly 5x106 cells per dish (10 cm) or in total if non-adherent. Rinse each dish
briefly with ice cold PBS to remove medium.
- Then add 2 ml of cold methanol and scrape cells from plate. Wash plate with 3 ml of cold
PBS. Pool in screw cap tubes (13x100 mm with teflon ring).
- Add 4 ml chloroform and vortex vigorously.
- Spin for 5 min in clinical centrifuge at 2000rpm. If phase separation is not apparent, add a
few grains of NaCl and repeat the spin at room temperature.
- Of the lower chloroform phase use 2.5 ml for the DAG/ceramide assay and 0.8 for the
total lipid phosphorus. Dry lipids down under a stream of nitrogen. In this form they can be
stored at -20C for up to 48 hours.
2. Solubilization of dried cellular lipids for phosphorylation of DAG/ceramide by E.coli
Diglyceride kinase (DGK).
- DGK is somewhat particular in terms of how its substrate must be presented to get
phosphorylated (see Walsh & Bell, 1986, JBC 261: 6239-6247). In a mixed micellar system
the best combination is 10 mM phosphatidylglycerol (PG), diC 18:1 (diC18:0 also works)
solubilized in 7.5% beta-octylglucoside (b-OG) with 1 mM diethylenetriamine pentaacetic
acid (DTPA).
- For 100 l of this solution dry down 32.05 l PG under nitrogen, add 100 l of 7.5% bOG and 0.2 l 500 mM DTPA. Each cellular lipid sample should be solubilized in a no
more than 50 l of this solution. To facilitate dissolving lipids sonicate gently. The solution
must appear clear.
3. Phosphorylation of DAG/ceramide using DGK present in membranes from E. coli
overexpressing the protein.
- Make 2X reaction buffer with the following composition100 mM imidazol pH 6.6, 100
mM NaCl, 25 mM MgCl2, 2 mM EGTA.
- Make labeling stock for the equivalent of 10 samples as follows: 500 l 2x reaction
buffer, 10 l 100 mM cold ATP (final conc. should be 2 mM) and 10 l of E. coli
107
membranes containing DGK. Preincubate for 30 min. to phosphorylate endogenous DAG

present in the E. coli membranes (N.B. these membranes do not contain ceramides). Then
add 10 l of 32P-gATP (at least 3000 Ci/mmol).
- Per lipid sample (50 l volume) add 50 l of this labeling mix and incubate at room
temperature for 30 min.
COMMENTS: from the moment hot ATP has been added, everything must be done in the
B-lab
4. Extraction of phosphorylated reaction products, phosphatidic acid (PA)
and ceramide phosphate (Cer-P).
- Stop reaction after 30 min. by adding 3 ml methanol:chloroform (2:1) and 0.7 ml 1% HCl.
With 100 l initial aqueous volume PA should remain soluble in the chloroform phase due
to protonation by HCl.
- Then add 1 ml chloroform and vortex again. This will lead to the formation of two phases
and separation should occur. Add another 1 ml of 1% HCl and vortex again.
- Spin in clinical centrifuge for 5 min at 500 rpm. Aspirate away water phase, but do not
remove interphase!
- Wash twice with 2 ml 1% HCl (volume equal to chloroform phase), each time removing
upper phase after spin. After last spin remove all aqueous phase and interphase. Make all
sure all aqueous phase is gone, otherwise PA will be hydrolyzsed to lyso-phosphatidic acid
(LPA), which migrates in a different position.
COMMENTS: For these steps it is highly advantageous to work with 200mm glass screwcap tubes. This reduces significantly the danger of contamination during vortexing.
5. TLC analysis of samples
- For analysis between 200 l (sufficient for DAG analysis) and 1 ml (required for
ceramide analysis) of chloroform phase are dried down. Note that DAG levels are 5-10 fold
higher than ceramide levels.
- Dry sample down under nitrogen and resuspend sample in 50-100 l of
chloroform/methanol (4:1). Spot on a silica plate that has been divided into lanes and prerun in acetone. Develop in a TLC chamber containing chloroform/ pyridine/formic acid
(60:30:7) for about 2 hours.
- Dry plate in hood for at least 1 hour or use a fan to speed up drying process. Wrap plate in
Saran-wrap to seal in pyridine and expose plate overnight at RT to be able to locate the
appropriate spots for scraping next day.
- Scrape spots into 3 ml scintillation fluid and count.
COMMENTS: To speed up sample spotting use a hair dryer. To facilitate scraping moisten
silica matrix with a water sprayer.
108
DAG/CERAMIDE & PHOSPHATE WORKSHEET

DAG/CERAMIDE ASSAY:
DATE:
SAMPLES:
BLIGH & DYER:
used
ml methanol
ml chloroform
ml PBS
ml chloroform phase for DAG/ceramide

ml chloroform phase for phosphate assay
O immediately
O stored over night after drying down
PHOSPHATE: Standards
phosphate (nmol)
OD 820nm
P1
P2
P3
Regression analysis: y = ax + b;
a=
P4
P5
; b=
P6
; r=
LABELLING STOCK:(calculate per sample a requirement of 55 l stock)

l 2x reaction buffer
l 100mM cold ATP
l E. coli membranes
min. preincubation at RT
l
use
50
32
P-gATP
l per lipid sample

min. incubation at RT
EXTRACTION/TLC: Volume of HCl-washed chlorofom phase

Volume dried down under nitrogen
ml
ml
samples used O immediately

O stored over night
Volume to resuspend PA, Cer-P
Volume spotted onto TLC
l
l
VALUES FROM SCRAPING TLCs

Standards (ceramide/DAG)
S1
ceramide (nmol)
(cpm)
S2
109
S3
S4
S5
S6
a=
; b=
; r=
DAG (nmol)
(cpm)
a=
; b=
; r=
Lipid samples
cpm Cer-P
ceramide (nmol)
cpm PA
DAG (nmol)
OD 820nm
phosphate (nmol)
L1
L2
L3
L4
L5
L6
Lipid samples
cpm Cer-P
ceramide (nmol)
cpm PA
DAG (nmol)
OD 820nm
phosphate (nmol)
L7
L8
L9
L10
L11
L12
Lipid samples
cpm Cer-P
ceramide (nmol)
cpm PA
DAG (nmol)
OD 820nm
phosphate (nmol)
L13
L14
L15
L16
L17
L18
COMMENTS:
AQ, 15-2-96
110
ISOLATION OF DGK MEMBRANES

Two strains of DGK overexpressers are available:
N4830 with plasmid pJW10 (Loomis et al.,1985, JBC 260: 4091-4097),
DH5 with plasmid pKRdgk (Ramer and Bell, 1990, JBC 265: 16478-16483). The latter
strain has higher levels of DGK expression.
PROCEDURE
1. Grow E. coli over night in L-Broth
2. Spin in clinical centrifuge at 5000 rpm to collect pellet
3. Wash pellet in TNE buffer (50 mM Tris-HCl pH7.5, 0.1M NaCl, 1 mM EDTA, 5
mM -mercaptoethanol). Washed pellets may be stored at -20 C.
4. Resuspend 6g of packed E. coli in 294 ml 50 mM potassium phosphate pH7.5,
150 mM KCl, 50 mM sodium pyrophosphate, 5 mM EDTA, 5 mM mercaptoethanol.
5. Disrupt cells by passing them through a cold French Press 2x at 16000 psi. At
BIL there is no French Press, so try a combination of lysozyme treatment with
sonication as is used for the preparation of GST fusion proteins.
6. Unbroken cells are removed by centrifugation for 15 min at 5000xg.
7. Supernatant is spun at 200000xg for 2 hours.
8. Pellet is resuspended in 150 ml TNE buffer and spun again for 1 hour at
200000xg.
9. Pellet is resuspended in phosphate glycerol buffer (10 mM phosphate pH 7.0,
20% glycerol) to a final concentration of 1 mg protein/ml and frozen away in
aliquots at -20C.
10. Per DAG/ceramide assay 1-5 g of membrane protein are employed.
COMMENTS
AQ, 15-2-96
111
LIPID PHOSPHORUS DETERMINATION

(Ames and Dubin)
MATERIALS
Block heater 160C (Ismatec Labor Technik AG)
45C water bath
70% perchloric acid (Fluka #77230)
0.9% ammonium molybdate (Fluka # 09878), store at room temperature wraped in foil
1mM potassium dihydrogen phosphate (0.0136g/100ml, store at 4C)
10% ascorbic acid (Fluka #95209) prepared fresh
PROCEDURE
1. Dry down samples under N2 gas
2. Prepare standards by pipetting 0-60 nmol (0-60l of potassium hydrogen
phosphate) into tubes
3. Add water to 300l and add 150l perchloric acid to all tubes
4. Heat in 160C block heater (in fume hood) overnight
5. Add 700l double destilled water (nano-pure)
6. Add 500l 0.9% ammonium molybdate, mix well
7. Add 150l 10% ascorbic acid, mix, incubate at 45C for 30 min.
8. Read absorbance at 820nm
COMMENTS
Standard block heaters only go up to about 140C. This should work anyway. You
can tell the lipid has been hydrolysed if it appears clear, as apposed to slighlty
yellowish.
Do not let your samples dry out in the block heater, since dry perchloric acid is an
explosive.
The bottom limit on the assay is about 3-4 nmols
112
LIPOSOME ASSOCIATION ASSAY

(centrifugation assay in airfuge)
Buffer:50 mM HEPES pH8.0
PS (Mw 810) 25 mg/ml= 30mM
10 mM MgCl2
PC (Mw786) 25 mg/ml =33.3mM
1 mM EGTA pH7.5-8 DiC8(Mw 344)25mg/ml=72.7mM
5 ug/ml BHT
Ceramide
0.1 mM DTPA
PA
PIP
PIP2
PC:PS
PC:PS
volume ratio
molar ratio
per 200ul liposome stock:
(500 ug/ml)
3 : 1 ul
(3.6 mg/ml)
22: 7 ul
160 ul
400 ug/ml
20 ul
400 ug/ml
liposomes
assay
Ca
(ug/ml)
per assay volume of 180 ul:

final lipid concentration:
fusion
protein
protein vol. per

conc.
PDBu
(1mM) (1 uM)
1/
2/
3/
4/
5/
6/
7/
8/
9/
10/
11/
12/
Notes:
AQ, 15-6-96
113
INHIBITION OF DYNAMIC PROTEIN PALMITOYLATION IN INTACT CELLS

WITH TUNICAMYCIN
By SEAN I. PATTERSON and J. H. PATE SKENE
Introduction
Biological roles of protein palmitoylation have been investigated principally
through mutational analysis of individual protein substrates.1-4 An important limitation of
this approach is its inability to examine dynamic aspects of palmitoylation. This approach
also is confined to analysis of individual, cloned protein substrates and by the availability
of appropriate cell systems for expression of the mutated proteins. Availability of a specific,
cell-permeable inhibitor of protein palmitoylation could complement the mutational
approach and would greatly facilitate investigation of the involvement of dynamic protein
palmitoylation in various cellular functions. Such an inhibitor would permit observation of
the effects of acute disruption of ongoing protein palmitoylation in living cells or
subcellular fractions of interest.
The only documented inhibitor of protein palmitoylation in intact cells has been the
antibiotic cerulenin.5,6 However, it has not been established whether cerulenin can serve as a
general inhibitor of cellular protein palmitoylation, and there is evidence that at least some
examples of dynamic palmitoylation are not sensitive to the inhibitor.3-7 Furthermore, as a
covalent modifier of protein cysteine thiols, 8 cerulenin is known to inhibit synthesis of fatty
acid, phospholipid, sterol, protein, and RNA as well as protein acylation, 8-11 and there are no
clear criteria for distinguishing cellular effects that may result from disruption of protein
palmitoylation from those attributable to other actions of cerulenin. We have shown that the
fatty acid containing nucleoside antibiotic tunicamycin (TM, see Fig. 1) can inhibit
palmitoylation of at least some proteins that are resistant to cerulenin. Palmitoylation of
several proteins in neuronal cell cultures, including the identified growth cone substrate
GAP-43,12 was not affected by cerulenin, but it was rapidly and reversibly inhibited by
TM.13 Tunicamycin also is known to inhibit protein glycosylation (see Table I). Here we
describe experimental criteria for distinguishing the effects of TM on intact cells that can be
attributed specifically to its ability to inhibit posttranslational protein palmitoylation.
TABLE 1
CHARACTERISTICS OF EFFECTS OF
PALMITOYLATION
AND GLYCOSYLATION
Paramter
Palmitoylation
Time of onset
_10 min
Dose range
1-25 g/ml
Reversibility
_ 15 min
Posttranslational effects
Yes
Structural dependence
Chain length
Effective in serum
No
Fatty acid reversibility
Yes
a
A. M. Heacock, Brain Res. 241, 307 (1982).
TUNICAMYCIN
ON
PROTEIN
Glycosylation
Possible lag of 30-60 mina
0.05-5 g/ml
Several hours
No
No systematic effect
Yes
No
The conceptual bases for distinguishing the effects of TM on protein glycosylation

and palmitoylation arise from differences in the mechanisms of TM inhibition of the two
protein modifications and the degree of dependence on protein synthesis. Tunicamycin
inhibits N-linked glycosylation by acting as a transition-state analog for the enzyme UDPGlcNAc:dolichyl phosphate GlcNAc-1-phosphate transferase.14 In contrast, structural
comparison suggests that TM may inhibit protein palmitoylation by competing for binding
114
of the lipid donor palmitoyl-CoA to the protein fatty acyltransferase. 13 The models predict
that TM inhibition of palmitoylation, but not its effects on glycosylation, should be
attenuated by increasing intracellular levels of palmitoyl-CoA. Furthermore, the ability of
different homologs of TM (Fig. 1) to inhibit palmitoylation should reflect the known
preference of protein palmitoylating enzymes for longer chain fatty acids. A second basis
for distinguishing TM effects on glycosylation and palmitoylation is that N-linked
glycosylation is tightly linked to protein synthesis in the endoplasmic reticulum and near
Golgi compartments.14,15 Palmitoylation, in contrast, occurs both cotranslationally and with
a subsequent posttranslational component that occurs at the cell membrane. 16,17 Protein
synthesis inhibitors, therefore, can be used to disrupt N-linked glycosylation and the
cotranslational component of palmitoylation, without directly affecting the more dynamic
posttranslational protein palmitoylation.
FIG. 1. Structure of tunicamycin showing the variation in fatty acyl side chains for the
different homologs. The uracil and tunicamine moieties remain the same in the homologs,
whereas the number of carbons in the acyl side chain varies from 14 (A series) to 17 (D
series), with additional variations in the branching and saturation as shown.
Probing Cellular Functions of Palmltoylation with Tunicarnycin
In the absence of a more specific inhibitor of palmitoylation, experimental criteria
derived from the differences discussed above make it possible to use TM to investigate the
functional roles of dynamic protein palmitoylation in intact cells. In previous work, for
example, we used TM to probe the role of protein palmitoylation in the process of neuronal
growth cone motility.13 Tunicamycin caused a rapid and reversible collapse of motile
growth cones that correlated with its ability to inhibit protein palmitoylation but not
glycosylation. Similar application of TM should be useful for investigating the involvement
of protein palmitoylating enzymes in a variety of other cellular activities. In practice, the
distinction between TM effects on palmitoylation and glycosylation is most clear when the
process being investigated is measured over short time periods, generally in the range of a
few minutes to 2 hr. This minimizes the confounding effects of TM inhibition of
glycosylation and permits the use of protein synthesis inhibitors as a control for TM actions
on cotranslational events.
We have observed the inhibition of protein palmitoylation by TM in several cell
types, including dorsal root ganglion (DRG) neurons, cortical neurons, PC12
pheochromocytoma cells, 3T3 fibroblasts, and L cells. This effect of TM is thus likely to be
115
widely applicable to many cell types. However, certain biochemical aspects of the
inhibition should be reinvestigated in any new cellular system in which TM is to be used to
probe the functional roles of protein palmitoylation: (i) rapid inhibition of the
posttranslational incorporation of [3H]palmitate into cellular proteins at moderate doses
(typically <25 g/ml), without significant disruption of palmitoyl-CoA synthesis or
incorporation of palmitate into cellular lipid; (ii) preferential inhibition of palmitoylation by
long-chain TM homologs; (iii) reversal or attenuation of that inhibition in the presence of
increased extracellular palmitate levels, which can transiently increase intracellular levels
of palmitoyl-CoA. For systems in which they apply, these features of TM inhibition,
together with protein synthesis inhibitors, can be used to explore the functional
consequences of acutely inhibiting dynamic post translational protein palmitoylation.
Assaying Functional Correlates of Protein Palmitoylation Using Tunicamycin
A stock solution of TM appropriate for use in many functional assays can be
prepared by dissolving commercial preparations of TM, or constituent homologs, in 10 mM
aqueous sodium hydroxide at 1 mg/ml. Small amounts of particulates can be removed with
0.43-m filters (Millipore, Bedford, MA, type HV). If a substantial amount of material
remains undissolved (especially after freeze/thaw), discard and make up with fresh sodium
hydroxide solution. Dilute 10 l into 0.99 ml culture medium and measure the absorbance
at 260 nm against a medium plus sodium hydroxide solution blank. The absorbance of 10
g/ml TM should be 0.12 absorbance units. Most buffered culture media will tolerate
considerable quantities of vehicle (up to 10% by volume) without substantial effect. Salts
can be added to the stock solution to reduce the osmotic shock to cells, although calcium or
magnesium in the stock solution can precipitate TM.
An important consideration in selecting conditions for functional assays is that high
concentrations of serum strongly attentuate the inhibition of protein palmitoylation by TM.
The mechanism by which this occurs is not clear, although it is possible that uptake of free
fatty acids or lipoproteins elevate intracellular levels of fatty acyl-CoA. Regardless of the
mechanism, it is important in practice to carry out functional assays in serum-free or lowserum conditions.
FIG. 2. Incorporation of extracellular [ 3H]palmitate into DRG neuron lipids. Cultured DRG
neurons (1 ganglion per sample, <104 cells) were labeled with 10 M [3H]palmitate (600
Ci/ml) for 30 or 60 min. Aliquots of lipids equivalent to 1/10 of the total extract were
analyzed by TLC as described in the text, with phase partitioning. The lower and upper
phases from the partitioning are shown at left (organic) and right (aqueous). Radioactivity
was measured by 5-day fluorographic exposure to preflashed x-ray film at -70C. The
migration positions of free fatty acids (FFA) and phosphatidylcholine (PC, left) and
palmitoyl-CoA (PalmCoA, right) are indicated.
It should be confirmed that labeling of the protein(s) of interest under given assay
conditions reflects dynamic posttranslational attachment of palmitic acid to cysteine thiols,
as described previously12,13,18 and elsewhere in this volume. 19-21 It is also important to
demonstrate that functional responses to TM are correlated with an inhibition of the protein
116
palmitoylation, measured by incorporation of [ 3H]palmitate. We have observed that

incorporation of [3H]palmitate into protein and/or lipid can saturate abruptly after
incubations as short as 30 min, although in general labeling has been linear up to 60 min.
Observations of protein palmitoylation and its dependence on different labeling conditions
are thus most reliable when carried out as time courses in the range of 5-60 min, although
this can quickly become unrealistic in terms of materials and analysis. However, we
routinely measure the incorporation of radioactivity into lipid (Fig. 2), as described below,
to control for inter- and intraexperimental variability, and as a measure of the metabolic
health of cells exposed to known cytotoxic conditions.13-22
Functional responses to TM can be rapid. Disruption of growth cone morphology
and of neurite extension by cultured DRG neurons, for example, are apparent within 3-10
min of TM addition at concentrations in the range of 1-10 g/ml.13 Measuring inhibition of
protein palmitoylation within this time frame is technically arduous in intact cells, owing to
the time required for uptake of radiolabeled fatty acid and metabolic conversion to
palmitoyl-CoA, and for sufficient incorporation of the labeled palmitate to permit detection
and quantitation. Labeling times of 20-60 min nevertheless can show whether protein
palmitoylation is inhibited effectively under the experimental conditions of the functional
assay. If a temporal correlation at short times is necessary, the onset of TM inhibition of
palmitoylation can be estimated by regression analysis of the inhibition of labeling at
longer time points (Fig. 3). Such analysis suggests, for example, that the onset of inhibition
occurs within 5 min of addition of 10 g/ml TM to PC12 cells.
FIG. 3. Time course of the onset of TM inhibition of posttranslational protein

palmitoylation. A series of cell cultures were labeled with [ 3H]palmitatc in the presence of
100 g/ml cycloheximide for up to 40 min (open circles); at 20 min. 10 Lg/ml whole TM
was added to some cultures (arrow), and labeling was continued for an additional 20 min
(filled circles). Triplicate cultures were removed at 5-min intervals for analysis of
[3H]palmitate incorporation into the protein SNAP-25, and the results are plotted in
arbitrary densitometry units. Solid lines indicate the best-fit least-squares regression lines
for labeling in the presence and absence of TM.
Because TM appears to inhibit most or all posttranslational palmitoylation without
distinguishing between substrate proteins,13 TM disruption of a particular cellular activity
can indicate the involvement of palmitoylating enzymes but not the contributions of
individual palmitoylated substrates. One- or two-dimensional electrophoresis of
[3H]palmitate-labeled proteins at multiple time points can be used to screen for candidate
substrates that undergo turnover on the same time scale as the functional responses to TM,
or to demonstrate TM inhibition of palmitate incorporation into previously identified
proteins of interest. Attribution of a specific cellular role to the ongoing palmitoylation of
any individual protein will require a combination of TM inhibition studies with a
complementary approach, such as mutational analysis, specific for individual protein
substrates.
Purification Tunicamycin Homologs by High-Performance Liquid Chromatography
Once a cellular function or activity of interest has been shown to be inhibited by
TM, under conditions in which TM also inhibits posttranslational protein palmitoylation, it
117
is important to ascertain whether the cellular effects result from blocking palmitoylation or
from other actions of TM, including its well-known inhibition of glycosylation. The first
experimental criterion for making this distinction is preferential inhibition of the function of
interest by TM homologs containing long-chain fatty acids (Fig. 1). We have previously
shown that protein palmitoylation is inhibited preferentially by the C and D families of TM
homologs, which have fatty acyl side chains containing 16 and 17 hydrocarbons,
respectively.13 This feature can be exploited by comparing the functional effects of the
shorter and longer chain homologs, either individually or as families, at intermediate
concentrations, such as 2-5 g/ml. At those concentrations, homologs of the different
families are maximally and equally active in the inhibition of the N-linked glycosylation
pathway in intact cells.23
TABLE II
CONDITIONS FOR S~PARATION OF TUNICAMYCIN HOMOLOGS AND FAMILIES
BY HPLC
Condition
Method I
Method II
Separation
Individual homologs
Homolog families
Temperature
40
60
Flow Rate
1 ml/min
1 ml/min
Elution
Isocratic, 80 min
Gradient, 25 min
Equilibration Solvent
Methanol/water (68:32, v/v)
Methanol/water (72:28, v/v)
Loading
_0.5 mg
_1mg
Whole TM, containing a mixture of the naturally occurring homologs, can be
purchased commercially from a number of sources (Sigma, St. Louis, MO; Boehringer
Mannheim, Indianapolis, IN). As the composition of the whole TM with respect to the
individual homologs varies from batch to batch, different batches of TM will vary in the
potency with which they inhibit protein palmitoylation. To compare the abilities of different
TM homologs to inhibit cellular activities of interest, individual TM homologs can be
obtained commercially, or larger quantities of the homologs can be prepared more
economically by separation from commercial batches of whole TM. Two methods are
presented here for the separation of TM homologs (Table II). The first, based on the method
of Mahoney and Duksin,24 will separate all the homologs of known structure and activity 23;
the second is a related but more rapid method for the collection of the homologs as families
defined by the number of carbons in the side chains (Fig. 1), from A (14-carbon side chains)
to D (17-carbon side chains). Both methods require a high-performance liquid
chromatography (HPLC) system with a column temperature controller, and on-line UV
detection. In the examples shown here, we used a Waters (Milford, MA) 625 system with a
temperature control module and the RCM-100 column heater, and a 994 programmable
photodiode array detector. There are many suppliers of the reversed-phase C8 columns used
for the homolog separation; here we use the Rainin (Emeryville, CA) Dynamax 4.6 x 250
mm Microsorb Cat. No;~0-325-C5) with a guard column (Cat. No. 80-300-G5), which we
have found to give highly reproducible results over many separations. It is particularly
helpful to wash the column with 100% methanol between runs, and with methanol/water (3:
7, v/v) at 60 at the end of each day of separations.
TABLE III
HPLC PROFILES FOR ELUTION OF INDIVIDUAL TUNICAMYCIN
HOMOLOGS OR HOMOLOG FAMILES
Time
Eluanta
Gradient
Method 1
0-70 min
68% (v/v) methanol
Isocratic
70-80 min
100% (v/v) methanol
Isocratic
Method 2
0-3 min
72% (v/v) methanol
Isocratic
3-15 min
72-78% (v/v) methanol
Linear
118
15-25 min
78-100% (v/v) methanol
Linear
Whole TM is dissolved in HPLC-grade methanol/water (7:3, v/v) at 1-5 mg/ml,

filtered (Millipore type HV, 0.43 ,um), and up to 0.5 mg (method 1) or 1 mg (method 2)
loaded onto the column. High yields can be rapidly obtained by separating the families with
method 2 prior to separating the individual homologs with method 1, described in Table III.
It should be noted that the elution times of the separated homolog families are slower by 35 minutes than when running whole TM. The separated C and D homolog familes can also
be collected with an isocratic elution of 72% methanol to keep the run times under 40 min,
although care must be taken not to overload the column with the higher methanol elution.
Alternatively, collection of the homologs as families of the same chain-length from the
separation by method 2 alone yields purities of _90%. This quick separation may be
sufficient for comparing short and long-chain homologs for activity.
The column effiuent is monitored at 260 nm (Fig. 4), and peaks may be collected
individually or as 1-min fractions and subsequently pooled after reference to the chart. The
fractions should be collected in glass tubes, and all subsequent handling or transfer steps
should be carried out in glass or with glass pipettes. The longer chain C and D families of
homologs are particularly prone to adsorption to polypropylene or polystyrene tubes,
especially after drying. Loss of material through adsorbtion to the containers can still be
substantial with the less abundant short-chain TM homologs. Pooled fractions of individual
homologs or homolog families are then taken to dryness in a Speed-Vac concentrator
shielded from the light. When stored dry and in the dark at -20 to -80, the homologs are
stable for prolonged periods.
FIG. 4. Representative separations of whole TM into individual homologs (method 1) or

homolog families (method 2). TM (Boehringer Mannheim, Lot No. 12838820-20) was
injected through a 2O0-l loop onto a Dynamax C8 reversed-phase column (4.6 X 250 mm)
equilibrated and run as described in the text and in Tables II and III. The eluant was
monitored on-line at 260 nm. Automatic acquisition of spectra (200-400 nm) for eluting
peaks confirmed their identity as genuine TM.
Attenuation of Tunicamycin Effects by Exogenous Fatty Acid
A second and more conclusive criterion for attributing functional effects of TM to
its inhibition of protein palmitoylation is protection by increased levels of extracellular
palmitate. The rationale is that palmitic acid in the medium is taken up by cells and
converted to the acyl donor palmitoyl CoA, at least transiently elevating the intracellular
concentration of the donor with which TM must compete to inhibit palmitoylation.
Although the competitive model of TM inhibition has not been established, we have found
empirically that high levels of extracellular palmitate can in practice attenuate.the ability of
TM to inhibit protein palmitoylation and can reverse TM-induced inhibition of neurite
growth. Because exogenous fatty acid does not interfere with TM inhibition of N-linked
glycosylation, this criterion can distinguish functional effects attributable to TM inhibition
of protein palmitoylation from those due to inhibition of glycosylation.
119
The uptake and intracellular metabolism of palmitic acid are rapid and subject to
complex modulation. Additionally, cells have a large capacity to buffer intracellular acylCoA levels.25 Palmitoyl-CoA is rapidly metabolized, and intracellular regulatory
mechanisms will tend to restore palmitoyl CoA levels after a transient rise in response to
increased uptake of the fatty acid. For this reason, incubations with high concentrations of
fatty acid should be kept to a maximum of 1 hr, and preferably shorter. Because exogenous
palmitic acid produces only a limited and transient increase in intracellular palmitoyl-CoA,
the ability of extracellular palmitate to reverse functional deficits induced by TM will be
most apparent at subsaturating doses of TM.
Care should be taken in the preparation of the cell culture medium and other
solutions containing high concentrations of palmitate, as palmitic acid at the concentrations
required to reverse TM inhibition (10-100 M) tends to precipitate in the presence of
millimolar concentrations of divalent cations. Fresh solutions should be made by diluting
the fatty acid 1000-fold from stock solutions in dimethyl sulfoxide (DMSO), followed by
brief sonication in a bath sonicator. If the solutions are cloudy, they should be discarded and
a new solution made at a slightly lower concentration of fatty acid. In this regard, it should
be borne in mind that serum may serve as a source of fatty acids and lipoproteins that can
attenuate TM inhibition of palmitoylation. 13 Conversely, proteins such as bovine serum
albumin (BSA) will bind fatty acids and sequester them from the cells, as well as binding
TM and reducing its effectiveness as an inhibitor (Table IV). Solubilizing agents such as hydroxypropylcyclodextran will also avidly sequester TM and are inappropriate for drug
delivery.
TABLE IV
ALBUMIN REVERSIBILTY OF CELLULAR PROTEIN PALMITOYLATION AND
TUNICAMYCIN INHIBITION
3
BSA
H-Palmitoylation of SNAP-25a
Inhibition
(g/ml)
Control
10 g/ml TM
(% control)
0
2190 209
642 76
71
0.1
1771 453
708 12
60
1
1282 175
909 189
29
10
291 33
282 132
3
Arbitrary densitometric units.
Inhibition of Protein Synthesis with Cycloheximide
Protein N-linked glycosylation is tightly linked to protein synthesis (Fig. 5),
whereas a substantial component of protein palmitoylation is independent of translation.
One can therefore use protein synthesis inhibitors, alone and in combination with TM, to
distinguish further cellular responses to TM that can be explained by the inhibition of Nlinked glycosylation from those that may reflect functional roles of dynamic
posttranslational protein palmitoylation.
Most nonmitochondrial cellular protein synthesis can be stopped by treatment with
the cell-permeable drug cycloheximide. As there are variations in cellular sensitivity,
determination of a suitable dose for each new cell type is recommended in the absence of
prior knowledge. Protein synthesis can be monitored by the incorporation of
[35S]methionine or cysteine (for 20-60 min with 50-500 Ci/ml) after pretreatment of the
cells for up to 60 min with cycloheximide in the dose range of 1-100 g/ml. Proteins can
then be analyzed individually after separation on polyacrylamide gels, or in bulk after
precipitation with trichloroacetic acid. Once an effective dose of cycloheximide has been
determined, one can conduct parallel labeling experiments with [ 3H]mannose and
[3H]palmitate to confirm that N-linked glycosylation is effectively inhibited, and to
determine what proportion of cellular protein palmitoylation is posttranslational. In our
experience, the relative amount of palmitoylation that is independent of protein synthesis
varies for different cell types and for different protein substrates within each cell, which
120
likely reflects both differential rates of synthesis and differential turnover of the palmitate
moieties for individual proteins.
Because it inhibits N-linked protein glycosylation (Fig. 5), cycloheximide or other
protein synthesis inhibitors should mimic cellular responses to TM attributable to its
inhibition of glycosylation, or to a partial inhibition of protein synthesis that occurs in many
cells.23 In contrast, functions that depend on posttranslational palmitoylation can continue
for relatively long periods of time in the absence of protein synthesis. In cultured DRG
neurons, we have found that growth cone motility and active neurite extension continue
unabated for over 2 hr in the presence of 100 g/ml cycloheximide, whereas PC12 cell
growth cones remain active for 60-90 min. Incorporation of [ 3H]palmitate can be used to
determine how long palmitoylation of cellular proteins in general, or of individual proteins
of interest, persists in the absence of protein synthesis in other cellular systems. In
functional assays, prolonged persistence of an activity in the presence of cycloheximide,
contrasted with rapid disruption on treatment with TM, provides evidence that the effect of
TM is distinct from the inhibition of protein synthesis or glycosylation and may be due to
disruption of protein palmitoylation.
FIG. 5. TM and cycloheximide both inhibit protein glycosylation. PC12 cells

differentiated with nerve growth factor13 were labeled with [3H]mannose for 2 hr with no
addition (Control), 20 g/ml TM (TM), or 100 g/ml cycloheximide (Cyclo) added
concurrently. Proteins were precipitated with TCA and separated by 12% SDS-PAGE, as
described in the text. Tbe incorporation of radioactivity was determined by a 2-month
fluorographic exposure to X-ray film at -70. Arrows indicate the migration positions of
molecular weight standards (x10-3) of (from top to bottom) 106, 80, 49.5, 32.5, 27.5, and
18.5.
A further use of cycloheximide is to determine whether functional properties
disrupted by TM can recover after washout of TM in the continued presence of the protein
synthesis inhibitor. Recovery of functional defects resulting from depletion of glycosylated
proteins should depend absolutely on de novo protein synthesis. This is not true for shortterm recovery from inhibition of posttranslational protein palmitoylation.
Inhibition of Protein Palmitoylation in Isolated Preparations
In some cases, it may be valuable to study functional involvement of ongoing
protein palmitoylation in events that are most readily assayed with isolated membrane
fragments, organelles, or other subcellular preparations. It is necessary in those cases to
confirm that the preparation of interest retains the relevant acylating and/or deacylating
enzymes, that active palmitoylation continues under the assay conditions, and that TM can
effectively inhibit palmitoylation under those conditions.
A number of preparations have been described for the cell-free assay of protein
palmitoylating activities.13,17,26,27 Isolated preparations, especially lysed membrane
preparations, may not retain ATP and acyl-Co-A synthase, soluble factors required to
121
convert radiolabeled palmitic acid to palmitoyl CoA, the immediate donor for protein
palmitoylation. In the case of neuronal growth cones, however, we have found that a
protein palmitoylating activity remains tightly associated with extensively washed
membrane preparations.13 It is likely that other subcellular preparations also retain protein
palmitoylating activities, expression of which will require addition of exogenous palmitoylCoA, either directly or by providing free fatty acid, coenzyme A, and an exogenous acylCoA synthase. Unlabeled palmitoyl-CoA is available commercially. Labeling studies to
measure active protein palmitoylation, and its inhibition by TM, in such isolated
preparations require radioactive palmitoyl-CoA, which is available commercially but can
be prohibitively expensive in the amounts required for biochemical studies. We therefore
provide a simple method for the preparation and isolation of [3H]palmitoyl-CoA from
commercially available supplies of [3H]palmitate. In using TM as an inhibitor of growth
cone-derived acyltransferase activity, we found it necessary to use high concentrations (up
to 1 mg/ml) of the antibiotic in the presence of micromolar levels of [3H]palmitoyl CoA. In
the presence of 17 nM [3H]palmitoyl-CoA, however, TM was an effective inhibitor in a
similar range to that effective on intact cells.13 Unfortunately, incorporation of radiolabel
into protein at that concentration required long fluorographic exposures (2 months) for
reliable quantitation.
Experimental Procedures
Assay of Tunicamycin Inhibition of Posttranslational Protein Palmitoylation
1. To inhibit protein synthesis, a suitable concentration of cycloheximide (from a
stock solution of 10 mg/ml in phosphate-buffered saline) should be added to cell cultures 1
hr prior to labeling, and to the wash and incubation solutions.
2. Wash cell monolayers (subconfluent in 6- or 24-well dishes, or equivalent) or
suspended cells at least twice with serum-free culture medium containing no more than
0.01% (w/v) fatty acid-free bovine serum albumin (BSA) at 37. If bicarbonate-buffered
medium is used, a suitable buffer (e.g., 20 mM HEPES) should be included to prevent
medium alkalinization in the absence of CO2.
3. Commence cell labeling by adding culture medium as in Step 2, but containing 3
M [3H]palmitic acid (DuPont NEN, Boston, MA, 60 Ci/ mmol) and cycloheximide, or by
adding the radiolabel from a concentrated stock in DMSO (only for palmitate
concentrations of less than 10 M, keeping the final DMSO concentration below 0.1% by
volume).
4. Quickly add TM stock solution (in 10 mM NaOH) or vehicle alone, and agitate
gently to mix. Cell monolayers can be examined briefly under a microscope to ensure that
no precipitation of TM or fatty acid has taken place.
5. Incubate at 37 for between 5 and 60 min.
6. Terminate the labeling by the addition of 10 volumes of chloroform / methanol
(1:1, v/v) to suspended cells. With attached cells, the labeling medium should be carefully
removed with a pipette, transferred to a polypropylene vial, centrifuged at 14,000 g at 4
for 3 min, and the supernatant removed. The attached cells should be scraped into
methanol, pooled with any pelleted material from the labeling supernatant, and adjusted to
chloroform/methanol/water (5:5:1,v/v/v). For a zero-time control, quench unlabeled cells
and then add a suitable quantity of radiolabel to the organic solvent mix.
7. Extraction of lipids and precipitation of proteins should be allowed to continue
for 30 min on ice. For small amounts of protein (<5 g) precipitation overnight at -20 is
helpful.
8. Pellet precipitated proteins by centrifugation (14,000 g for 15 min at 4).
9. Transfer a measured aliquot of the supernatant and allow to evaporate at 37 for
at least 2 hr, before taking to dryness in a Speed-Vac concentrator. This prevents the
samples from boiling over when the vacuum drops below 10 millibar. This sample is used
for lipid analysis by thin-layer chromatography (TLC), as described below.
122
10. Remove and discard the remaining supernatant. For large pellets repeat the
organic wash 1-3 times with centrifugation to remove residual label. Redissolve the
proteins in sample buffer [1% sodium dodecyl sulfate (SDS), 20% glycerol, 5 mM
dithiothreitol (DTT), 0.025% (w/v) bromphenol blue, 10 mM Tris-HCl, pH 7.4) for analysis
by SDS-polyacrylamide gel electrophoresis (PAGE) and fluorography, as described
elsewhere.20,28
11. An alternative to PAGE analysis is to assess the incorporation of radiolabel into
total cellular protein after labeling with [3H]palmitate as described, or with [ 3H]myristate
(DuPont NEN, 30 Ci/mmol) at 50-100 Ci/ml (higher concentrations may be cytotoxic).
After Step 8 the samples are repeatedly washed with chloroform/methanol/water (5:5:1,
v/v/v), with centrifugation between each wash. An aliquot of each wash is dried and
counted in a scintillation counter until the extraction of radioactivity levels off (usually 3-6
washes). Two further washes are then made with chloroform/methanol (2:1, v/v). The pellet
is redissolved in 1% SDS/10 mM Tris (pH 7.4) with heating, and the radioactivity is
determined in a scintillation counter.
Preparation of [3H]palmitoyl-CoA
1. Dry 100-1000 Ci [3H]palmitate (60 Ci/mmol, DuPont NEN) in a glass screwcapped vial, and redissolve in 100 l of 0.05% Triton X-100, 1 mM CoA (Boehringer
Mannheim),5 mM ATP, 5 mM MgCl2, 10 mM Tris (pH 7.5) containing 1 U/ml acyl-CoA
synthase (Boehringer Mannheim). It is helpful to dissolve the fatty acid prior to the addition
of MgCl2 from a 100x stock.
2. Incubate at 37 for 1-2 hr with occasional gentle vortexing.
3. Add 1 ml chloroform/methanol (1: 1, v/v) and mix thoroughly.
4. Add 0.5 ml chloroform and 0.275 ml water, vortex for about 30 sec, and then
centrifuge briefly to obtain a clean separation of the phases. Recovery of radioactivity in
the upper aqueous phase should be 50-90%, and analysis by TLC (as below) should show at
least 90% radiochemical purity.
Analysis of [3H]Palmitate-Labeled Lipids
1. Lipid extracts, prepared as described above, are redissolved in
chloroform/methanol (2:1, v/v) and spotted onto aluminum-backed silica gel 60 TLC plates
(Art. 5553, EM Science, Gibbstown, NJ) cut to a height of 10 cm.
2. Heat the plates briefly (100-110 for _5 min, do not go over 115), and after
allowing to cool develop with butanol/acetic acid/water (5: 2: 3, v/v/v) 29 in an enclosed
preequilibrated TLC tank for approximately 8 cm.
3. Remove the plates from the TLC tank and allow to air-dry in a fume hood.
4. For samples with low amounts of radioactivity, the plates can be permeated with
2,5-diphenyloxazole (10%, w/v, in ether) for fluorography. Pour the solution over the tilted
plate in the direct of the developing, as some smearing of the neutral lipid and fatty acid
will occur (see Fig. 2). The dried TLC plates are then exposed for 3-10 days at -70 to Xray film (X-OMAT AR, Eastman Kodak, Rochester, NY) preflashed to an optical density of
0.1 absorbance units. After development, radiolabeled bands are quantitated by
transmittance optical density and identified by reference to labeled lipid standards (see
above), or unlabeled lipid standards visualized by exposure to iodine vapor.
5. In intact cells, the incorporation of label into material comigrating with
phosphatidylcholine is often great enough to overwhelm the signal from palmitoyl-CoA. If
this is the case, aliquots of lipid extract can be enriched for palmitoyl-CoA by phase
partitioning. Redissolve the dried lipid extracts in 1.1 ml chloroform/methanol/10 mM Tris,
pH 7.4 (5: 5: 1, v/v/v), then add 0.5 ml chloroform and 0.275 ml water and split the phases
as described above for the synthesis of [3H]palmitoyl-CoA. Transfer the phases and dry
separately in a Speed-Vac concentrator. The upper aqueous phase will be substantially
enriched in palmitoyl-CoA (see Fig. 2).
123
Measurement of Protein Synthesis and Glycosylation

1. Cells are prepared and incubated under the same conditions as for the relevant
protein palmitoylation experiment, but instead of [3H]palmitate, [35S]cysteine (Amersham,
Arlington Heights, IL, 50-500 Ci/ml) or [3H]mannose (DuPont NEN, 50 Ci/ml) are used
as the radiolabel.
2. After the appropriate labeling period, incubations are quenched by the addition of
an equal volume of 20% (w/v) ice-cold trichloroacetic acid (TCA), and the samples are
placed on ice for 60 min.
3. Cells grown on monolayers are then scraped and transferred to polypropylene
vials.
4. Insoluble material is pelleted (14,000 g for 15 min at 4) and the supernatant
discarded.
5. The pellet is washed with the same volume of 1% (w/v) TCA, and then twice
with 1/10 volume ether (presaturated with water), with centrifugation as above in between
each wash.
6. Redissolve the pellet in a minimum volume of 100 mM Tris-buffered 1% SDS
(pH 7.4) with heating.
7. Determine incorporation of radioactivity by counting in a scintillation counter
after addition of an appropriate water-tolerant scintillant.
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M. E. Linder, C. Kleuss, and S. M. Mumby, this volume [25].
21
D. R. Pepperberg, D. F. Morrison, and P. J. O'Brien, this volume [28].
22
D. T. Hess, S I. Patterson, D. S. Smith, and J. H. P. Skene, Nature (London) 366, 562
(l993).
23
D. Duksin and W. C. Mahoney, J. Biol Chem. 257, 3105 (1982).
24
W. C. Mahoney and D. Duksin, J. Chromatogr. 198, 506 (1980).
124
25
A. Arduini, G. Mancinelli, G. Radatti, S. Dottori, F. Molajoni, and R. Ramsay, J. Biol.

Chem. 267, 12673 (1992).
26
T. Yoshimura, D. Agrawal, and H. C. Agrawal, Biochem. J. 246, 611(1987).
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M. F. G. Schmidt and G. R. Burns, Biochem. Soc. Trans. 17, 625 (1989).
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D.E.Garfin,this series,Vol.182,p.425.
29
H. Juguelin and C. Cassagne, Anal Biochem. 142, 329 (1984).
125
LONG-CHAIN FATTY ACYLATION OF PROTEINS

Sean I. Patterson and J. H. Pate Skene
1. Introduction
Reversible modifications of cellular proteins often serve as key steps in signal
transduction and effector pathways, and the investigation of these modifications has been a
principal route of access to signaling mechanisms. Ten years ago, for example, tyrosine
phosphorylation was an intriguing odditya posttranslational modification known to occur
in virtually all eukaryotic cells and to be regulated by various manipulations that alter cell
growth or differentiation, but with no clear physiological role. From the elucidation of
tyrosine kinase pathways emerged a dramatically new view of how cells transduce and
integrate signaling cascades controlling cellular functions from growth to death (see
Chapter 6).
Today, another dynamic posttranslational modificationthe covalent attachment of
palmitate or other long chain fatty acids to cysteine residues (Schmidt, 1989; James and
Olson, 1990)is coming to be recognized as an important component of signal
transduction pathways. Like tyrosine phosphorylation a decade ago, S-palmitoylation today
is known to occur in all eukaryotic cells and to be regulated by a variety of signals that alter
cell growth, metabolism, or repair (Huang, 1989; James and Olson, 1989; Mouillac et al.,
1992; Hess et al., 1993; Wedegaertner and Bourne, 1994; Robinson et al.,1995). Recent
work has shown that palmitoylation is a dynamic process that can alter the targeting of
proteins to specific membrane domains (Peitzsch and McLaughlin, 1993; Silvius and
l'Heureux, 1994; Parton and Simons, 1995), the activation of signal transduction pathways
(Casey, 1995; Milligan et al., 1995), and the transforming activity of some oncogenic
proteins (Kato et al., 1992).
Long-chain fatty acylation is one of a growing class of lipid modifications of
proteins, which includes modifications by phospholipids, isoprenoids, and possibly
retinoids as well (Schmidt, 1989; Casey, 1995). Among these modifications, long-chain
fatty acylation stands out as the only one with a substantial posttranslational component
that includes active cycles of acylation and deacylation throughout the life-time of the
protein. Because this dynamic acylation continues after proteins have reached vesicles,
plasma membrane, and other cellular compartments, it is segregated spatially from other
types of lipid modifications that occur cotranslationally or during early protein processing
in the endoplasmic reticulum and Golgi.
In the nervous system, active cycles of protein palmitoylation and depalmitoylation
occur not only in neuronal cell bodies, but also in axon terminals (Skene and Virag, 1989;
Patterson and Skene, 1994) and myelin membranes (Bizzozero et al., 1987; Bizzozero and
Good, 1991). The functional and regulatory roles of this protein palmitoylation are only
beginning to be resolved, but available evidence already suggests that dynamic cycles of
palmitoylation play critical roles in neural development. Inhibition of ongoing protein
palmitoylation in axonal growth cones produces a rapid disruption of axon elongation,
implying a fundamental involvement of protein palmitoylation either in the constitutive
machinery for axon extension or in signaling pathways for axon guidance (Patterson and
Skene, 1994). More recently, characterization of the first enzyme involved in dynamic
protein palmitoylation, an extracellular protein depalmitoylating enzyme, revealed that
mutations in depalmitoylation may be responsible for one form of neuronal ceroid
lipofuscinosis, a severe neurological disorder characterized by early loss of visual function
and progressive mental retardation (Camp and Hofmann, 1993; Vesa et al., 1995).
Prominent and active palmitoylation of key proteins involved in myelin formation
(Bizzozero et al., 1987; Yoshimura et al., 1987; Pedraza et al., 1990) and synaptic
transmission (Hess et al.,1992; Gundersen et al., 1995) suggests that investigation of S126
palmitoylation and its regulation will provide valuable insights into signaling mechanisms
in the mature nervous system brain as well as in development.
S-palmitoylation refers to the posttranslational linkage of long acyl chains to
specific cysteine residues on target proteins through a relatively labile thioester bond. The
name refers to the thioester linkage and to the observation that the modifying acyl chain is
usually palmitate, a saturated 16-carbon fatty acid. Some other long-chain fatty acids may
be able to substitute for palmitate when they are available, but because palmitate is the most
abundant long-chain fatty acid in cells, it is likely to be the predominant fatty acid for longchain S-acylation of proteins under most cellular conditions. Although other long-chain
fatty acids may occasionally substitute for palmitate, shorter acyl chains are strongly
excluded from this reaction. The primary distinction occurs between acyl chain lengths of
16 and 14 carbons (Peitzsch and McLaughlin, 1993; Silvius and l'Heureux, 1994; Vogt et
al., 1994). This chain length specificity is one important difference between Spalmitoylation and a separate lipid modification, N-myristoylation. The 14-carbon saturated
fatty acid, myristate, is not used for posttranslational S-acylation of cysteine residues, but
can be attached cotranslationally in a stable amide linkage to amino terminal glycine
residues of appropriate substrate proteins (Grand, 1989; Schmidt,1989). N-myristoylation is
a critical processing steps for some proteins, but unlike S-palmitoylation, N-terminal
myristate does not undergo active cycles of posttranslational removal and replacement of
the lipid chain, a key requirement of modification reactions used for dynamic regulation of
cellular proteins. In addition to its biological importance, the existence of this Nmyristoylation reaction is a practical concern when using radiolabeled fatty acids to identify
protein substrates for S-palmitoylation, since palmitate may be converted metabolically to
myristate.
Although a large number of S-palmitoylated proteins have now been characterized,
remarkably little is understood about the enzymes and mechanisms involved in either the
attachment or removal of palmitate. Only one depalmitoylating enzyme has been isolated, a
secreted palmitoyl-protein thioesterase whose endogenous substrates remain unknown
(Camp and Hofmann,1993). Despite many attempts, no protein palmitoylating enzyme has
been isolated. There is, furthermore, no clearly defined recognition sequence for
palmitoylation, as in the case of other lipid modifications (Casey, 1995; Milligan et al.,
1995). For these reasons, some investigators have suggested that incorporation of longchain fatty acids may occur by chemical transfer from the activated acyl coenzyrne A (CoA) donor to a receptive thiol group, without involvement of a transferring enzyme.
Nonenzymatic chemical acylation has been demonstrated to occur in vitro (O'Brien et
al.,1987; Ouesnel and Silvius,1994). Although this matter will not be formally resolved
until the purification of a palmitoylating enzyme or demonstration of chemical transfer in
vivo, several investigators have described cell-derived protein-acylating activities (Berger
and Schmidt, 1984; Bizzozero et al., 1987; Yoshimura et al., 1987; Schmidt and Burns,
1989) that evince properties not easily reconciled with the chemical transfer model. These
include specificity for chain length of the acyl Co-A donor, sensitivity to heat and
enzymatic degradation, and preferential acylation of two adjacent cysteine residues.
Selective acylation of adjacent cysteine residues has been described in two S-palmitoylated
proteins, bovine opsin and GAP-43, and may represent a more general trend of dual
acylation by the same or different lipids (Ochinnikov et al., 1988; Liu et al., 1993). Such
dual acylation may prove to be of functional significance, since in vitro experiments
suggest that acylation at two or more sites will tether a protein to a specific membrane,
whereas single acylation allows rapid exchange between adjacent membranes (Shahinian
and Silvius, 1995).
Protein S-palmitoylation is beginning to emerge as an important modification for
dynamic regulation of proteins in many cells, including developing and adult neurons and
glial cells. In this chapter, we describe methods for labeling and analyzing palmitoylated
proteins in neurons, based on approaches originally described in the literature, which we
have adapted over several years. Naturally, readers may wish to adapt these procedures,
described in detail in the following sections, for their own specific applications. We have
127
therefore included descriptions of some of the problems encountered and some theoretical
background to facilitate a painless transfer of these procedures to different systems.
2. Reagents and Equipment
2.1. Sources of Specfflc Reagents
Most of the reagents described here are easily available from international suppliers,
such as Sigma (St. Louis, MO). [3H]Palmitic acid is available from a number of suppliers.
We have routinely used the product of DuPont/New England Nuclear (Boston, MA) at a
specific activity of 50-60 Ci/mmol, which is economically priced when purchased in 25mCi lots. [3H]Palmitoyl-CoA is also available commercially, but is pro hibitively
expensive. In Section 4.1.1., we describe a quick and cheap method for the synthesis of
[3H]palmitoyl-CoA from [3H]palmitate. Hydroxylamine-HCl, N-ethylmaleimide, and
cycloheximide can be obtained from Sigma. Whole tunicamycin and some of its purified
homologs are available from Boehringer Mannheim (Indianapolis, IN) and Sigma, or the
homologs can be purified by high-pressure liquid chroma tography as described (Patterson
and Skene, 1995). Organic s~3lvents should be of the highest available purity and at least
reagent grade.
2.2. General Equipment Requirements
Equipment required for the techniques described in this chapter include an aspirator
line with catchment bottle (for radioactive media), ice buckets, microfuge, centrifugal
vacuum desiccator (e.g., Savant Speed-Vac), rotary shaker, vortex, bath sonicator, heating
block or boiling water bath, one- and two-dimensional gel apparatus for the separation of
proteins, protein electroblotting apparatus, thin layer chro matography tank and plates,
autoradiographic film and developing facilities, autoradiographic cassettes, and the
facilities for handling and safely disposing of organic sol vents and of radioactive solids
and liquids.
2.3. Handling of Lipids and Solvents
A major cause of problems in handling lipids is the use of inappropriate containers
for storage or transfer. Lipids should routinely be kept in clean glass containers, sealed with
some organic-solvent inert cap, such as PTFE, or with Teflon coating. Although the extent
of the problem is not clear, palmitate will adhere to the polypropylene containers most
commonly used for labeling reactions, and this problem can become severe when working
with small amounts or in deter gent-free aqueous solutions. Therefore, when drying down
fatty acids or lipid extracts for redissolving in aqueous solu tions, glass containers are
always preferred. Regardless of the type of container, dried fatty acids to be resuspended in
aqueous media should be sonicated in a bath sonicator for 10 min after addition of the
aqueous buffer to improve recov ery. Organic solvents will usually redissolve dried lipids
efficiently from polypropylene containers without sonication, although it does not hurt to
take precautions. It is difficult to attain quantitative transfer of volatile solvents with air dis
placement pipets, so we recommend the use of positive displacement pipets for these
solvents whenever possible. ~-Never use polystyrene pipets or containers with organic
solvents. Many problems with handling lipids and organic sol vents arise from
contamination with finger grease or deter gents from improperly rinsed glassware. Rinsing
glassware with ethanol or acetone prior to use and using tubes from containers only
accessed with gloves can help prevent these problems.
3. Methodological Considerations
3.1. Approaches for Measuring Different Aspects of the Acylation Cycle
128
The most commonly used method for measuring pro tein acylation is the use of a
radiolabeled fatty acid tracer (e.g., [3H]palmitate), followed by extraction of the proteins
and analysis by gel electrophoresis and quantitative auto radiography. This is a
straightforward technique applicable to systems from isolated membranes to tissues in vivo.
The most substantial limitation is that the incorporation of label into pro teins occurs under
nonequilibrium conditions, so that measure ment of the incorporated radiolabel cannot be
converted to a quantitative estimate of the steady-steady mass or molar ratios of fatty acid
incorporated into a particular protein. Nonethe less, this is the technique of choice for
demonstrating active protein palmitoylation in a particular tissue, cell type, or mem brane
system, and for identifying palmitoylated proteins.
Among the problems that can be encountered in using [ 3H]palmitate incorporation
to measure protein acylation are the initial lag as the fatty acid is converted to its metaboli
cally active acyl-coenzyme A derivative, and the sometimes rapid and abrupt saturation of
labeling. The latter problem may result from either depletion of radioactive fatty acid or
saturation of some intermediate metabolic pool. This satu ration effect can confound the
comparison of protein palmitoylation observed under different conditions. One approach to
solving this problem is to compare the time course of palmitate incorporation under
conditions in which the incorporation of radiolabel into the protein(s) of interest is linear.
With this approach, both increased and decreased incorporation of [ 3H]palmitate can be
reliably observed, even at short times occluded by the lag in labeling (see, for example,
Fig.3 in Patterson and Skene [1995]) or other transient effects.
Another approach to measuring regulation of palmitoy lation is pulse-chase
incorporation of [3H]palmitate. This technique has been used successfully to demonstrate
receptor regulated deacylation of specific proteins (James and Olson, 1989; Wedegaertner
and Bourne, 1994). In our hands, this technique also suffers from problems arising from
satura tion when the original labeling is carried out under condi tions optimized for
maximum labeling. Like other fatty acids, radiolabeled palmitate is rapidly incorporated
into a broad range of cellular lipids, creating intracellular pools of radio labeled palmitate
that may confound quantitative analysis of depalmitoylation during a chase period. One
solution is to use the cell-permeable thiol-modifying agent N-ethyl maleimide to block
covalently deacylated cysteine thiols (Hess, personal communication) as described in
Section 4.2.1., step 5.
3.2. Criteria for the Identification of Thioester-Linked Long-Chain Acylation
Lipid modification of proteins extends well beyond the incorporation of long-chain
fatty acids to cysteine thiol groups. Proteins tethered to the extracellular surface by
glycophosphatidylinositol anchors frequently include palmi tate as their side chains (see
Chapter 11). Furthermore, meta bolic conversion of the abundant palmitate to the rare
cellular fatty acid myristate occurs rapidly, giving rise to overlap ping labeling of
palmitoylated and myristoylated proteins. Prolonged incubations can even result in labeling
of proteins through conversion of the [3H]fatty acid to [3H]acetyl-CoA, and incorporation of
the radioactivity into amino acids. It is therefore frequently necessary to demonstrate
directly whether labeling of proteins after incubation with [3H]palmitate represents Spalmitoylation or another protein modifi cation. This includes determination of the nature
of the chemical bond linking the labeled lipid to the protein and identification of the
attached lipid moiety.
3.2.1. Linkage Analysis
Fatty acids are known to be attached to proteins through both thioesters (to
cysteines) and oxyesters (to serines and threonines), and by amides and oxyesters in
phospholipids. Thioesters are particularly sensitive to nucleophilic attack by such agents as
hydroxylamine (Schmidt and Lambrecht, 1985), which under neutral conditions, pH 7.08.0, can be used as a diagnostic test to distinguish S-palmitoylation of cysteine residues
129
from the other forms of lipid attachment. Under more basic conditions, pH 10.0-12.0,
removal of fatty acid is more rapid and quantitative, but oxyesters also become susceptible
to cleavage. The common way of ana lyzing protein-lipid linkage is to separate
[3H]palmitate labeled proteins in two identical samples on two parallel lanes of a
polyacrylamide gel (see Chapter 3), and then to assess the loss of incorporated radiolabel
after incubating one of the gel lanes in neutral hydroxylamine:
1. Disassemble the gel apparatus and fix the gel for 30 min 25% (v/v) isopropanol in water
at room temperature with gentle agitation. Wash the gel in several changes of water
(210 gel volumes) for 30-60 min (shorter times for lower-percentage acrylamide gels).
2. Divide the lanes and place one in at least 10 gel volumes 1.0M hydroxylamine HCl
(Sigma) made to pH 7.0-8.0 KOH. Place the second gel piece in an equal volume 1.0M
Tris-HCl made to pH 7.0-8.0 with KOH. Incubate at room temperature for 4 h with
gentle agitation.
3. Wash the gel pieces with water as in step 1.
4. Stain and destain the proteins in the gel, for example, .05% (w/v) Coomassie brilliant
blue in isopropanol/ H2O/acetic acid 25:65:10 to control for differential loading of
proteins or loss during incubations.
5. Prepare the gel for autoradiography or fluorography (as described in Section 4.1.3., step
15).
3.2.2. Fatty Acid Identification
Cellular proteins can incorporate a number of different fatty acids, generally
segregated into short (12-14 carbon acyl chains) and long (>16 carbon acyl chains) fatty
acylation. Techniques exist for the quantitative analysis of endogenous fatty acids within
cellular proteins, but they are laborious, require specialized equipment, and for many
purposes are excessive. The analysis of incorporated fatty acid is routinely carried out after
labeling with radiolabeled myristate or palmitate to identify each kind of modification.
However, since cells are capable of converting each label into the other (Olson et al., 1985),
formal demonstration that the incorpo rated radioactivity represents in large part the same
fatty acid as added is required. The techniques for fatty acid analysis presume that sufficient
radiolabeled protein (about 10-fold the amount used for simple [3H]palmitate incorporation
into protein analysis) canbe acquired in a reasonably pure form that is separated from
other radiolabeled proteins. Ideally, this can be carried out by polyacrylamide gel
electrophore sis, although for rare proteins or when the migration posi tion of the protein on
a gel is crowded or ambiguous more exhaustive purification is called for.
1. Separate the [3H]palmitate labeled proteins on an appropriate polyacrylamide gel,
remove from the apparatus, and fix in three changes of 25% (v/v) isopropanol in water,
with gentle agitation for a total of 3 h. Using prestained markers or a reversible protein
stain to identify migration position of the relevant protein, cut out the part of the gel
containing the protein.
2. Dehydrate the gel fragment with three washes in at least 10 vol methanol for 60 min
to overnight.
3. Treat the gel fragment with 10 vol 0.1M KOH in methanol for 4 h at room
temperature.
4. Transfer the liquid to a separating funnel and extractwith 1 vol chloroform and 1 vol
H2O. Collect the lower(organic) phase and re-extract the upper (aqueous) phase -- with
an equal volume of chloroform. Pool the organic extracts and wash three times with
equal volumes of chloroform/methanol/H20 (1:10:10).
5. Dry the washed organic extract in a Speed-Vac or under a nitrogen stream in a glass
tube, redissolve in methanol, and spot onto Cl8 reversed-phase TLC plates (Whatman,
Hillsboro, OR), with methyl-fatty acid standards (saturated and unsaturated, C14-Cl8,
130
Sigma) in a parallel lane for comparison. Develop plates twice with acetone/metha
nol/H2O (8:2:1), drying thoroughly between the runs.
6. The migration position of the radioactive fatty acid(s) can be identified either by direct
autoradiography or by spraying the plate with a 10% (w/v) solution of 2,5 diphenyl
oxazole (PPO) in ether followed by fluorogra phy for lower levels of radioactivity. The
thoroughly dried plate should be apposed to suitable X-ray film (e.g., Kodak X-OMAT
AR) preflashed to an optical density of 0.1 and exposed at-70C. Marking the plate with
fluorescent paint can help to align the fluorogram after development.
7.To visualize the standards, separate the standard lane, spray with a 10% (w/v) solution
of phosphomolybdic acid (Sigma) in ethanol, air-dry, and then briefly heat to ~150C.
The standards should turn a dark, dirty green against a background that varies from
yellow to light green.
3.3. Measurements of Lipid Radiolabeling
The metabolic incorporation of radiolabel into proteins occurs through the same
intermediate used for the synthesis of lipids, that is, acyl-CoAs. The fraction of label added
to intact cells that ends up in protein is very small compared to the incorporation into
neutral- and phospholipids. Routine measurement of lipid-labeling confers several
advantages. The health and metabolic activity of the biological sample can be monitored,
since a robust incorporation of radioac tivity into the lipids demonstrates that the cells were
capable of taking up the fatty acid, converting it to the activated acyl CoA in an ATPdependent step, and finally incorporating the fatty acid into distinct lipid classes. The
pattern of lipid labeling can be quite distinctive and, thus, can be used to assess the purity
of the biological preparation, particularly with regard to contamination of cultures by
microorganisms. Bacteria appear to take up and incorporate [3H]palmitate into protein and
lipid more efficiently than mammalian cells and can thus give misleading results, even
when barely detect able by visual inspection of the cultures. If anomalous pro tein and
lipid-labeling patterns appear together, some form of contamination is quite likely.
The relative incorporation of [3H]palmitate into protein and lipid appears to vary
with the labeling conditions, and further with the metabolic state of the cells. It therefore
can not be reliably used to normalize results quantitatively between experiments. However,
quantitation can often provide useful information when changes in the incorporation of
[3H]palmitate are observed between samples within an experiment. Thus, parallel changes
in the level of protein and lipid-label ing more likely indicate variation in sample size or an
altera tion in the uptake or metabolism of fatty acid than direct alterations in the rate of
palmitoylation or depalmitoylation.
The starting point for the analysis of cellular lipids described here is the dried
organic extract of the labeling reac tion, as described in Section 4.1.3. (step 10).
1. Lipid extracts are redissolved in chloroform/methanol (4:1) and spotted onto aluminumbacked silica gel 60 TLC plates (Art. 5553, EM Science) cut to a height of 10 cm.
2. Heat the plates briefly (100-110C for <5 min; do not go over 115C); after allowing to
cool, develop once with butanol/acetic acid/H2O (5:2:3) in an enclosed pre-equilibrated
TLC tank for 8-9 cm.
3. Remove the plates from the TLC tank and allow to air-dry in a fume hood. Using a hair
drier can expedite this step.
4. For samples with low amounts of radioactivity, the plates can be permeated with 2,5diphenyloxazole (PPO) in ether for fluorography. Pour an ice-cold 10% (w/v) solution of
PPO in ether over the tilted plate in the direction of chromatography, since some
smearing of the neutral lipid and fatty acid will occur; then quickly dry with an air
stream or hair drier.
5.Expose the dried TLC plates for 3-10 d at -70C to X-ray film (Kodak X-OMAT AR)
preflashed to an optical den sity of 0.1 AU. After development, radiolabeled bands can
be quantitated by transmittance optical density and identified by reference to labeled or
131
unlabeled lipid stan dards. The latter can be transiently visualized by brief expo sure to
iodine vapor in a sealed tank (in the fume hood). The phosphomolybdate stain described
in Section 3.2.2. can also be used, but is not compatible with fluorography.
The incorporation of [3H]palmitate into material comi grating with
phosphatidylcholine is often great enough to overwhelm the signal from palmitoyl-CoA. If
this is the case, aliquots of lipid extract can be enriched for palmitoyl-CoA by phasepartitioning prior to step 1. Redissolve the dried lipid extracts in 1.1 mL
chloroform/methanol/10 mM Tris, pH 7.4 (5:5:1) with brief sonication. Then proceed as
described in Section 4.1.1., step 4. The upper aqueous phase obtained from this procedure
will be substantially enriched in pal mitoyl-CoA. This procedure is very sensitive to the
quantity of lipid and the salt content of the sample and may require considerable work to
establish reliably on a routine basis.
3.4. The Use of Protein Synthesis Inhibitors and Tunicamvcin
In many instances, it is important to distinguish palmito ylation during the initial
processing of a protein from ongo ing cycles of acylation and deacylation that may
subserve more dynamic roles in the regulation of protein localization or activity.
Cycloheximide and homologs of tunicamycin may be used to distinguish early processing
from the late post translational component of S-palmitoylation and to probe the functional
roles of ongoing palmitoylation in living cells (Patterson and Skene, 1994, 1995).
Cycloheximide inhibits pro in synthesis and, thus, the cotranslational component of all
forrns of protein acylation, without directly interfering with S palmitoylation. Furthermore,
primary neuronal cultures and many cell lines canbe pretreated with cycloheximide for at
least an hour before addition of radiolabeled palmitate without compromising cell viability
or metabolism. This effectively isolates the late posttranslational component of
palmitoylation and makes it possible to measure addition and turnover of palmitate separate
from early protein processing. For the special case of proteins in mitochondria, where
cotranslational palmitoylation may continue in the presence of cycloheximide, puromycin
or chloramphenicol also may be added.
Tunicamycin, a well-characterized inhibitor of protein glycosylation, also inhibits
posttranslational protein palmi toylation and, thus, can be used to probe the function of this
modification in intact cells (Patterson and Skene,1994). Because tunicamycin produces an
acute disruption of ongoing cycles of protein palmitoylation, it can be used to investigate
the functional roles of ongoing cycles of protein palmitoylation. Experimental procedures
and technical considerations in using these agents to investigate protein S-palmitoylation
have been described in detail elsewhere (Patterson and Skene, 1995).
4. Incorporation of [3H]Palmitate into Proteins
The techniques for labeling proteins with [3H]palmitate can be loosely divided into
two categories based on the form of the radiolabel: use of the immediate precursor of Spalmitoylation, palmitoyl-CoA, or use of palmitic acid and rely ing on metabolic
conversion to the active derivative (Table 1). Conceptually, this can be compared to the use
of 32P-labeled ATP or inorganic phosphate, respectively, for the phospho rylation of cellular
proteins. In both cases, the energetically activated donor of the protein-modifying group is
not cell permeable and can be used only in those systems in which access to the
intracellular compartment is provided, whereas the cell-permeable precursor requires
energy-dependent metabolic activation and is incorporated into multiple cellu1ar pools that
require separation from the target proteins of interest before analysis.
Table 1
Comparison of S-Palmitoylation with Palmitate or Palmitoyl-CoA
Label
Addition
S-palmitoylation of GAP-43a
132
2 M [3H]palmitoyl-CoA
100
ATP/CoA
139 6
3
2 M [ H]palmitate
86
3
2 M [ H]palmitate
ATP/CoA
71 37
200 M palmitate
93 9
3
2 M [ H]palmitoyl-CoA
200 M palmitoyl-CoA
74
a
Measured with 10-min incubations with growth cone membranes as described in the text
and expressed as a percentage (SD) of labeling with 2 M palmitoyl-CoA with no
additions.
4.1. S-Palmitoylation with [3H]Palmitoyl-CoA
The labeling of subcellular fractions, such as microsomes or membranes, either in
their native state or solubilized with detergent, provides important information about the
spatial segregation of palmitoylating reactions in the cell that may reflect the varying
functions of the substrates, and can be used to analyze factors impinging on the process in a
manner not available in intact cells. Whereas these components can be analyzed separately
by the use of protein synthesis inhibitors (Section 3.4.), palmitoylation in other fractions,
such as in the terminals of long neuronal axons or in the deter gent-resistant complexes that
may be specialized for signal transduction are not as easily distinguished.
The patterns of palmitoylated proteins in intact cellular vesicles labeled with
[3H]palmitate and membranes derived from these vesicles labeled with [3H]palmitoyl-CoA
are remarkably similar (Patterson and Skene, 1994). The palmito ylation of endogenous
proteins in membrane and membrane derived preparations assumes their copurification
with the membranes. It is sometimes desirable to use added proteins to follow the process,
to control their levels, or if the protein o~interest does not copurify with the membranes in
the pro tocol used. Furthermore, addition of a protein that has a free cysteine thiol, but does
not undergo S-palmitoylation in situ, is a useful control for the extent of nonenzymatic
palmi toylation under the specific reaction conditions used. Thus, we have used deacylated
preparations of the protein GAP 43 and bovine serum albumin (BSA) in conjunction to
optimize labeling conditions of the former over the latter. Section 4.1.2. includes a
description of the procedure for chemically reducing the thiols to ensure quantitative
control over the concentration of free protein thiol in the reaction solutions.
4.1.1 . Synthesis of [3H]Palmitoyl-CoA
1. Dry 100-1000 Ci [3H]palmitate (60 Ci/mmol, DuPont NEN) in a glass screw-cap vial
(e.g., Kimble Opticlear, 12 x 35 mm), and redissolve in 100 L of 0.05% (v/v) Triton X100, 1 mM CoA (Boehringer Mannheim, Mannheim, Germany), 5 mM ATP, 5 mM
MgCl2, and 10 mM Tris, pH 7.5, containing 1-10 U/mL long-chain acyl:coenzyme A
transferase (Boehringer Mannheim). Vortex gently but thoroughly to dissolve the fatty
acid while avoiding foaming. Add the MgCl2 from a 100-1000X stock after dissolving
the [3H]palmitate to avoid solubility problems with the fatty acid.
2. Incubate at 37C for 1-2 h with continuous gentle agitation or occasional gentle
vortexing; try to avoid foaming. If preferred, the reaction can be monitored by removing
microliter aliquots, diluting in 1.1 mL chloroform/methanol/water (5:5:1), and separating
the phaseswith chloroform and water as described in step 5. Small aliquots of the upper
(total ~0.75 mL) and lower (total ~1.13 mL) phases can then be transferred, dried in
scintillation vials (important to remove the chloroform that quenches the scintillants),
mixed with a scintillation cocktail, and the radioactivity determined in a scintillation
counter to calculate the conversion efffciency.
3. To quench the reaction, add 1 mL chloroform/methanol (1:1) and mix thoroughly,
preferably with brief bath sonication.
4. Spin down any precipitated material (5 min in a cooled microfuge at maximum speed)
and transfer the cleared supernatant to a fresh glass vial.
133
5. Add 0.5 mL chloroform and 0.275 mL H20, vortex for ~30 s or bath sonicate, and then
centrifuge briefly to obtain a clean separation of the phases.
6. Carefully remove the upper phase, without bringing any interface material or lower
phase, and transfer to another glass vial. Dry this material completely in a Speed-Vac
(~1-2 h) and redissolve in a small volume of dimethyl sulfoxide (DMSO).
7. Transfer aliquots (2 x 1 ,uL) to 1.5-mL microfuge tubes containing 0.5 mL methanol and
serially dilute 10- and 100-fold. Place the tubes in scintillation vials, add 10 mL
scintillant, and determine the radioactivity in a scintillation counter to calculate the
recovery.
8. A further aliquot (~1 L) should be diluted in chloroform/methanol for the analysis of
composition by TLC as described in Section 3.3.
Recovery of radioactivity in the upper aqueous phase should be 70-90%. Analysis
by TLC should show _90% radioactivity comigrating with S-palmitoyl-CoA standard
(Boehringer Mannheim), and the rest with free palmitic acid close to the solvent front.
An alternative technique for those who do not wish to involve themselves in
synthesizing label is to use the metabolic synthesis of [ 3H]palmitoyl-CoA in the
preparation. Membranes (and also detergent-solubilized membranes) con tain an
endogenous activity that will convert free fatty acids to their CoA derivatives in the
presence of ATP, CoA, and MgCl2. For membranes that do not contain a strong long chain
acyl CoA synthetase activity, the purified enzyme can be added. We have used this property
to synthesize actively and continuously [3H]palmitoyl-CoA from [3H]palmitate in labeling
mixtures by including 5 mM ATP and 1 mM CoA in the reactions. This method has the
advantage of continuously -replacing the [3H]palmitoyl-CoA that is consumed by lipid
synthesizing pathways. The disadvantage is that the endog enous palmitate will
immediately start competing with added radiolabel, rendering the specific activity of the
[3H]palmitoyl-CoA unpredictable. Thus, monitoring the level of [ 3H]palmitoyl-CoA in the
reaction will not provide informa tion on the chemical concentration of palmitoyl-CoA. In
side-by-side comparisons, we have found that either [3H]palmitoyl-CoA or [3H]palmitate
with ATP and CoA at equal concentrations could give more efficient radiolabeling of
proteins (Table 1).
4.1.2. Preparation of Reduced Protein Substrates for Palmitoylation
Proteins stored in aqueous solutions can undergo chemical oxidation of free cysteine
thiols, which interferes with subsequent quantitation of protein S-palmitoylation.
Fortunately, much of this oxidation is inter- or intraprotein formation of cystine bridges
(particularly with adjacent cysteines) that can be reversed by treatment with an appropriate
reducing agent, such as dithiothreitol (DTT).
1. Dissolve the protein(s) in a buffered aqueous solution suitable for subsequent dilution in
the labeling buffer.
2. Adjust to 10 mM DTT by addition from a 1M stock (which can be stored in aliquots at
-20C) and incubate at 55C for ~15 min.
3. Centrifuge the tube(s) in a microfuge for 5 min at maximum speed. If there is any
precipitate, a separate determination of actual protein concentration should be made(this
should not be a problem with freshly prepared proteins). Transfer supernatant aliquots to
the reaction tubes.
4. If nonreducing conditions are desired for the labeling reaction, proteins should be
reduced with DTT in advance, dialyzed against several changes of buffer at 4C, and can
be stored frozen in aliquots. In this case, do not reuse tubes after thawing.
4.1.3. Labeling of Membranes with [3H]Palmitoyl-CoA
134
Protein palmitoylating activity appears to remain strongly associated with

membrane preparations from many sources and probably represents an integral membrane
protein(s). Although different conditions are able to modify the efficiency of protein
palmitoylation with added radiolabel, overall the membrane-associated activity is robust
and tolerant to very different conditions. We have observed efficient labeling under
conditions varying from the most simple buffers (2 mM HEPES at pH 7.4) to complex
buffers that mimic the intracellular environment (see Table 2) or elution conditions from
ion-exchange columns.
1. Prepare suspended membranes at a concentration of ~1 /mL membrane protein (this can
be varied substantially according to availability or need).
2. Add 11X assay buffer (see Table 2). Add the appropriate amount of DTT and reduced
BSA stock solutions and aliquot the mix into screw-cap reaction tubes.
3. Add GAP-43 (or other protein) substrate if required. Add assay buffer to a final volume
of 100 L, or for time-courses (n + 1) x 100 L, where n is the number of time-points.
4. Transfer tubes to 37C and preincubate 10-15 min to allow the temperature to stabilize.
5. Initiate labeling by addition of [ 3H]palmitoyl-CoA (1-10 Ci/100 L _ 0.25-2.5 M)
from a stock solution in DMSO and mix immediately. Then return the tube to 37C.
Alternatively, [3H]palmitate (1-100 Ci/100 L), CoA, ATP, and MgCl 2 can be used as
described in Section 4.1.1.
6. For a time-course, remove an aliquot (100 L) before addition of label, transfer to a tube
containing 1 mL chloroform/methanol (1:1), and add an appropriate amount of
radiolabel to the quenched sample for the zero time point. Transfer further 100-L
aliquots to individual tubes at the desired times and quench with chloroform/ methanol
(1:1).
7. For single time-point experiments, quench the labeling by addition of 1 mL
chloroform/methanol (1:1) to each tube, followed by vortexing. Sonicate the tubes in a
bath sonicator for 15 min, and then continue the extraction at room temperature for at
least 30 min.
8. If the protein pellet did not break up into a smooth suspension in step 7, we recommend
continuing the extraction overnight at-20C. Precipitation of small amounts of protein
can be improved by transfer of the tubes to -20C for 30 min to overnight. This is not
usually necessary in the presence of added protein (e.g., BSA), which acts as carrier
protein.
9. Pellet proteins by centrifugation in a microfuge at top speed for 5 min at 4C. To prevent
contamination of the microfuge by radioactivity, it is important that all mixing and
centrifugation steps with organic solvents be carried out with tightly fitting screw-cap
tubes with some form of seal (e.g., rubber, Teflon, PTFE).
10. Transfer 110-L aliquots (i.e., one-tenth total volume) of the organic supernatants to a
parallel set of glass tubes, and warm to 37C for ~1 h without caps. Then take to dryness
in a Speed-Vac. The predrying period is to reduce the chloroform that boils over at the
low pressures of the Speed-Vac. Alternatively, ensure that the vacuum does not drop
below 5 mbar.
11. Remove the remaining supernatants from the protein pellets, using a gel loading tip to
get the final liquid from the bottom of the tube. Remember that this extract contains
most of the added radioactivity and that volatile solvents easily form contaminating
aerosols and drip from air-displacement pipets. We perform this step using a lowpressure aspirator with a "hot" collecting bottle.
12. Resuspend the pellets in 0.5 mL chloroform/methanol (2:1) and transfer to fresh tubes
with a further 0.5 mL wash (use positive displacement pipets or aerosol-resistant pipet
tips). Break up the pellets with vortexing and sonication as for steps 7 and 8. Centrifuge
as for step 9, and discard the radioactive supernatant. Repeat the organic wash with 1 mL
chloroform/methanol (2:1) without transferring the pellet to fresh tubes.
13. Allow the pellets to air-dry for ~30 min.
135
14. Add sample buffer (1% [v/v] SDS, 5 mM EDTA, 10 mM DTT, 20% [v/v] glycerol, 10
mM Tris-HCl, pH 7.0), and heat until the pellet is redissolved.
15. Separate the proteins by one- or two-dimensional gel electrophoresis as appropriate;
stain and destain as usual. Prepare the gels for fluorography by incubating in APEX
(55% [v/v] acetic acid, 15% [v/v] ethanol, 30% [v/v] xylenes, 1% [w/v] PPO) for 60 min
to overnight, followed by at least three 10-min washes in water. Dry the gel and appose
to preflashed X-ray film at -70C. Good exposures depend on the protein of interest, but
as a rough guide, they can be obtained within 1 wk to 1 mo.
4.1.4. Labeling with Solubilized Protein Acylating Activity
As mentioned above, a limiting factor in the analysis of protein palmitoylating
activities is the presence in mem branes of other metabolic pathways that also use
[3H]palmi toyl-CoA as a substrate, predominantly lipid synthetic pathways. One approach
to dealing with this limitation is the separation of the proteins responsible by any number of
techniques currently available, most of which first require solubilization of the proteins. We
have previously reported (Patterson and Skene, 1994) the solubilization of a protein
palmitoylating activity from the washed membranes of neu ronal growth cones using the
nonionic detergent Lubrol-PX (currently available under the tradename Thesit, from
Boehringer Mannheim), and a number of other groups have also successfully solubilized
similar activities with other deter gents. Distinct properties of these crude activities suggest
that there may be a family of protein palmitoylating enzymes.
Table 2
Solution for Palmitoylation of Membranes
Component
Final Concentration
Comments
NaCl
20 mM
From 11X stocka
KCl
100 mM
From 11X stock
CaCl2
0.1 mM
From 11X stock
MgCl2
1 mM
From 11X stock
HEPES, pH 7.1-7.2
20 mM
From 11X stock
BSA (fatty acid-free)
20 g/mL
Freshly reduced with DTTb
DTT
1 mM
From 1M stockc
3
d
[ H]palmitoyl-CoA
~0.3 M
From _100X DMSO stock
a
The salts and buffer can be made up as a single 11X stock solution, stable at room
temperature.
b
As described in the text for monitoring nonenzymatic acylation.
c
Aqueous solution aliquoted and stored at -20C.
d
Higher concentrations give progressively worse problems with chemical labeling.
The procedures we have used for protein palmitoylation proteins with detergentsolubilized membranes are essen tially the same as for the membranes themselves (Section
4.1.3.). The labeling reactions are carried out in a volume of 100 L at a protein
concentration of 1 mg/mL in the solution described in Table 2. The concentration of
[3H]palmitoyl-CoA is the same, as are the time-course, quench, and processing. The major
difference is that a smaller aliquot of lipid is used for the analysis by TLC to prevent
overloading of the TLC plate owing to the presence of detergent in the organic extract. It
should be noted that although the presence of detergent helps to suppress nonenzymatic
labeling of proteins (Patterson and Skene, unpublished data), it is worthwhile monitoring
this process routinely by addition of reduced BSA, as described in Section 4.1.2.
4.2. S-Palmitoylation with [3H]Palmitate
136
Labeling with [3H]palmitate shares the problems assoc iated with the metabolism of
[3H]palmitoyl-CoA, but has the advantage of not requiring direct access to the intracellular
compartment. It is thus useful for cultured cells, tissue slices, or even tissues in vivo,
although moving away from dispersed cells dramatically increases the problems of uptake
of radiolabel and competition with large endogenous pools of palmitate. Furthermore, the
extraction procedures for tissues are more laborious than for cultured cells.
The procedures for labeling require maintenance of the viable cells in a suitable
medium with [3H]palmitate. Unlike labeling with [3H]palmitoyl-CoA where concentrations
of label greater than low micromolar facilitate nonenzymatic labeling, it is desirable to use
high concentrations of extracellular fatty acid to compete as efficiently as possible with the
intracellular pools. The limitations encountered are thus physical (the solubility of the fatty
acid) or economic. In the presence of physiological concentrations of divalent cations, fatty
acids become insoluble between 10 and 50 M (the exact concentration depends on the
composition of the physiological salt solution). The presence of serum proteins can also act
as an alternative reservoir for fatty acid that will compete with the cells or tissue (Patterson
and Skene, 1994, 1995). When encountering failure to label proteins with [ 3H]palmitate,
solubility problems should immediately be considered as a possibility.
4.2.1. Labeling of Continuous Cell Lines and Primary Cultures
This section describes the procedure for labeling intact, cultured cells firmly
attached to a substratum, as developed for nerve growth factor-differentiated PC12 cells
and cultured adult dorsal root ganglion (DRG) neurons. The amounts and volumes
described below are for labeling of subconfluent PC12 cells or 2 DRG ganglia in a 35-mm
dish or 6-well plate.
1. Make up the physiological incubation medium. This is normally the same as culture
medium, but without serum or other fatty acid binding components. Bicarbonate
buffering should be used for incubation in a culture incubator. If incubations are to be
carried out without use of a humidified incubator, they should be kept short ('45 min) to
prevent substantial alterations in osmolarity resulting from evaporation. The medium
may also include cycloheximide at 10-100 g/mL in order to inhibit protein synthesis
(Section 3.4.). Incubation medium should be pre-equilibrated for temperature and gas
content, as appropriate.
2. -Dry down the desired amount of [3H]palmitate (plus a little extra for pipeting) in a glass
tube in a Speed-Vac. Redissolve in DMSO at 1000X the desired ffnal concentration.
3. Wash the cells with incubation medium. If the cells have been cultured with serum, then
at least two washes with an equivalent volume are required. If the cells were cultured
serum-free, then in principle the medium need only be replaced without washing. Take
care not to let the cells dry out, which dramatically reduces adhesion and viability. This
is particularly easy to do if the cells have been cultured without serum. Return the dishes
to the incubator for at least 15 min or at least 30 min if cycloheximide is being used to
inhibit protein synthesis.
4. To initiate labeling, add the desired amount of [3H]palmitate in DMSO. When operating
near the limit of solubility, it may be preferable to make up the labeling medium
separately with sonication in order to ensure good solubility. If this is not feasible, add
the label to the medium while swirling, but not so violently that cells are dislodged. With
phase-contrast microscopy, precipitated palmitic acid may be clearly seen as a fuzzy
contamination that tends to float to the medium surface. 5. If a chase is being performed,
remove the medium and either wash two times or replace with medium containing 0.1%
(w/v) fatty acid-free BSA + the relevant stimulant for chasing. At this stage, Nethylmaleimide (NEM) may also be included in the wash and chase buffer at.1-1 mM. If
cell adhesion is a problem, the medium may simply be replaced with medium containing
NEM. Allow 10 min before adding a chase stimulant. More than 30 min in the presence
of NEM can be highly detrimental to cell viability and adhesion. PC12 cells appear to be
137
much more sensitive to NEM treatment than primary cultures of adult sensory neurons,
and quite quickly lose adhesion with higher concentrations.
6. To terminate labeling, transfer the dish(es) to ice and wash with ice-cold medium with
sufficient EDTA to buffer divalent cations, or preferably with PBS/1 mM - --EDTA if it
causes no cell rupture or loss of cell adhesion. If there is loss of cell adhesion, the
original incubation medium should be transferred to microfuge tubes and pelleted (3 min
at 1000g at 4C), the medium aspirated, and the same tubes used to collect the scraped
cells from the equivalent wells or dishes (step 7). Furthermore, if BSA is used in a chase,
ensure that it is absent from the wash medium. Immediately after washing, quench the
cells by addition of 0.5 mL ice-cold methanol. At this point, either process the dishes
immediately, as described below, or store them at -20 or -80C until ready for
processing.
7. Scrape the cells into the methanol with a rubber policeman or a microspatula whose
angled end is covered with Tygon tubing (or some other inert tubing). Transfer the
suspension to screw-cap tubes (see step 6), and pool with a further 0.5-mL methanol
wash of each well or dish. The resulting volume is usually ~1 mL and can be assumed to
be ~0.75 mL methanol/0.15 mL wash medium. Add 0.75 mL chloroform to make up the
extraction solution of chloroform/methanol/water (5:5:1). Mix thoroughly with
sonication in a bath sonicator for 15 min.
8. Proceed with the analysis of protein and lipid as described in steps 8-15 of Section 4.1.3.
4.2.2. Labeling of Tissues In Vivo
Similar techniques to those described in the previous section can be used to label
proteins in tissue slices and neu rons in vivo (our own unpublished data, and data from
Hess [1992]). Although there is considerable information available about the
pharmacokinetics of [3H]palmitate in neuronal systems in vivo (Kimes et al.,1983,1985;
Tabata et al.,1986; Miller et al., 1987; Yamazaki et al., 1989), for labeling of proteins it can
briefly be summarized as a problem of delivering a suf ficient amount of label to the target
region. In vivo, only a fraction of injected radiolabeled palmitate ends up in protein,
resulting in a very low specific activity of the palmitate incorporated through Spalmitoylation in a comparatively large quantity of protein. There is also a delay in the
uptake of the radiolabeled fatty acid as it partitions in many physi ological pools, such that
longer labeling periods are required.
For the labeling of neural cells in the living rat retina, we (in collaboration with
Hess) have injected 2-4 L vol of a saturated solution of [3H]palmitate in DMSO (estimated
at about 1 mCi) into the aqueous humor of the eye of an anesthetized hooded rat using a
Hamilton syringe connected through inert tubing to a 28-gage needle inserted 3 mm
through the sclera anterior to the retina. The volume was chosen to minimize damage to the
neurons through increased pressure in the eye, and the solvent to be miscible with the
intraocular solution (to forestall catastrophic precipitation of the fatty acid in the presence
of the aqueous humor) and at a level nontoxic to neurons. The rats were permitted to
recover from anesthesia and were sacrificed at 4-24 h after the injection. The retinas were
dissected out, minced with a razor blade, and homogenized in a glass microhomogenizer in
chloroform/methanol/water (5:5:1). Analysis of the protein and lipid was then carried out
essentially as described in steps 8-15 of Section 4.1.3. The organic extracts of the
homogenized retina with chloroform/methanol (2:1) were monitored for radioactivity by
drying and scintillation counting, and the extractions were repeated until the radioactivity
extracted had leveled off close to background (three to six washes). The labeling of retinal
proteins could be seen at 4 h and peaked after 16 h injection.
5. Perspectives
A central characteristic of posttranslational palmitoy lation is the rapid replacement
of the fatty acid moieties compared to the degradation of the protein(s) to which they are
138
attached. It is not known which part of this cycle of attachment and removal is under
physiological regulation, or whether the primary functional significance is reflected most
directly in the rate of acyl chain attachment, removal, or the balance between the acylated
and deacylated forms. However, the most commonly used method for measuring
palmitoylation is the incorporation of externally added radiolabeled palmitic acid, under
conditions in which isotopic equilibrium has not been achieved. Although this is sufficient
to demonstrate that the protein(s) in question does indeed incorporate the fatty acid and to
proceed some distance in analyzing its regulation, it does not permit the direct
determination of any of these three variables required for quantitative analysis of the cycle
of palmitoylation in situ.
Many of the intracellular substrates for palmitoylation are known to be involved in
signal transduction and the control of cytoskeletal and membrane dynamics (Casey, 1995;
Milligan et al., 1995). The functional implications of palmitoylation of these proteins are
still unclear. However, it has been observed that palmitoylated proteins are enriched in a
specialized subcellular membrane fraction known as the detergent-insoluble,
glycosphingolipid-enriched complex (Parton and Simons, 1995, and unpublished data). In
many cells such domains are enriched in cell-signaling molecules, such as heterotrimeric
G-proteins and protein-tyrosine kinases, and in brain-derived synaptosomes they are
enriched in protein components of the synaptic membrane fusion machinery (Bennet et al.,
1992). Thus, palmitoylation may serve to concentrate components of signal transduction or
effector machinery within a small region of the membrane to increase the efficiency of their
interaction and promote directed cellular responses in a manner analogous to the
phosphorylation-dependent interaction of proteins containing SH2 domains.
The implication of this focus on specialized subdomains of the membrane is that the
tools we are currently using to investigate S-palmitoylation may still be too crude to extract
useful information about the functioning of the cycle in situ. Further technical
developments will be required before S palmitoylation can be measured with the kind of
quantitative exactitude currently possible with other protein modifying pathways.
Acknowledgments
Many of the techniques described in this chapter were developed with the help of D.
T. Hess (Department of Cell Biol ogy, Vanderbilt University, TN). We would further like to
thank him for sharing his own techniques prior to publication.
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plasma [14C]palmitate into the hypoglossal nucleus following unilateral axotomy of
the hypoglossal nerve in adult rat, with and without regeneration. Brain Res. 477,
19-28.
Yoshimura, T., Agrawal, D., and Agrawal, H. C. (1987) Cell-free acylation of rat brain
myelin proteolipid protein and DM-20. Biochem. J. 246, 611-617.
141
RECRYSTALLIZATION OF -OCTYLGLUCOSIDE
This is only necessary if the quality of octyl-b-D-glucopyranoside is not sufficient to

support DGK activity.
PROCEDURE
1. dissolve 12 g of beta-octylglucoside in 50 ml of freshly destilled acetone
2. Remove insoluble material by filtration
3. Add 200 ml of diethylether
4. Keep at -20C for 2 days
5. collect crystallized b-OG by filtration
6. Wash with 100 ml of diethylether at -20 C
7. Leave in hood to air dry
COMMENTS
Diethylether is highly explosive. Avoid close proximity of open flames.
AQ, 15-2-96
142
E: CELL BIOLOGY
ASTROCYTES ATTACHMENT ASSAY....................................................................144
ASTROCYTES ATTACHMENT ASSAY: IMMUNOBLOT OF PHOSPHORYLATED
PROTEINS....................................................................................................................146
AUROPROBE INDIRECT IMMUNOLOCALIZATION............................................147
COOMASSIEE BLUE STAINING OF MOUSE SPERM TO TEST ACROSOME
REACTION...................................................................................................................148
EFFECT OF ASTROCYTES ON EL-4 CELLS (ANTI-PY BLOT)............................149
IMMUNOFLUORESCENCE BY FACS ANALYSIS (IN PLATE ASSAY)...............151
IMMUNOFLUORESCENCE FOR CONFOCAL MICROSCOPY.............................152
INDO-1/FACS...............................................................................................................154
PY-IIF OF ASTROCYTES INCUBATED WITH EL-4 CELLS..................................155
PY-IIF OF ASTROCYTES INCUBATED WITH THY-1FC MOLECULES...............157
PY-IIF OF ASTROCYTES INCUBATED ON THY-1 COATED COVERSLIPS.......158
STAINING OF CELLS WITH CMFDA GREEN AND CMTMR ORANGE RED
(MOLECULAR PROBES)...........................................................................................160
STIMULATING EL-4 CELLS ON PLATES FOR ANTI-PY BLOTS.........................163
STIMULATING IL-2 PRODUCTION ON EL-4 CELLS............................................164
STIMULATING EL-4 CELLS TO PRODUCE IL-2 ON PLATES (OTHER).............166
TESTING ACROSOME REACTION IN HUMAN SPERM.......................................167
TREVORS FIXATIVE.................................................................................................169
(ALPHA-32P)GTP BLOT ANALYSIS..........................................................................170
ANTI-THY-1 BLOTS...................................................................................................171
EFFECT OF ASTROCYTES ON EL-4 CELLS (ANTI-PY BLOT)............................172
EXPRESSED PROTEINS PURIFICATION FROM HEK-293 SUPERNATANTS....174
LIGAND BLOT ANALYSIS........................................................................................175
PY BLOTS....................................................................................................................176
SPOT BLOT..................................................................................................................177
STIMULATION OF EL-4 CELLS (ANTI-PY BLOT).................................................178
IMMUNOPRECIPITATION.........................................................................................179
IMMUNOPRECIPITATION WITH LL95....................................................................180
143
ASTROCYTES ATTACHMENT ASSAY

- A maxisorp ELISA plate of 96 cm
- A 10cm plate with confluent astrocytes.
- Proteins to coat plate. All diluted in PBS
1Prepare plate folowing the template described in next page. Add 50 l per well.
Leave it overnight at 4C. Make sure liquid is well spread over the plate (tap
plate until even distribution)
2Wash once with PBS
3Block with 1% BSA in PBS. Add 200l/well and incubate for at least 2 hrs at
37C.
4Wash 3 to 4x with PBS
5Prepare the astrocytes:
Aspirate medium. Rinse 1x with PBS without Calcium and Magnesium
Detach cells with trypsin or trypsin EDTA treatment. Add trypsin solution at 37C
and wait for 1-2 min.
Collect cells disggregating with a pipette and add one volume of PBS/1mg/mlBSA.
Mix well
Wash them 2x with PBS and resuspend them at 2x105 cells/ml in PBS. For 4x106
cells add 20ml PBS, 250l of 2M glucose and 20l 0.1M MnCl2.
6Plate cells in wells. 200l/well (40 000 cells per well. This number depends on
the size of the cells. Cells should be enough to cover the well in only one layer).
Spin plate at 200rpm 4min. Incubate 1 hr at 37C
Note: this incubation can go upto 2 hrs but not longer.

7Wash gently once and check for cells in BSA wells. Usually one wash is
enough but, if cells are still left in BSA wells repeat the wash.
8Fix cells with 2-4% p-formaldehyde, leave at least 2 hr
9Wash once. Stain with crytal violet for 2-4hr (1%). Wash 1-2x with water.
Leave drying
10Measure at 620/580nm to quantitate the assay. Higher Absorbancy=higher cell
number.
TEMPLATE
144
RESULTS
145
ASTROCYTES ATTACHMENT ASSAY: IMMUNOBLOT OF PHOSPHORYLATED

PROTEINS
- A 6-well plate (Costar)
- 2x 10cm plate with confluent astrocytes.
- Proteins to coat plate. All diluted in PBS
1) Prepare plate folowing the template described. Add 1ml per well. Leave it overnight at
4C. Make sure liquid is well spread over the plate (tap plate until even distribution)
2) Wash twice with PBS
3) Block with 1% BSA in PBS. Add 100l/well and incubate for at least 2 hrs at 37C.
4) Wash 3 to 4x with PBS.
5) Prepare the astrocytes:
- Aspirate medium. Rinse 1x with PBS without Calcium and Magnesium
- Detach cells with minimum trypsinization treatment (1-2min). Add trypsin
solution at 37C.
- Collect cells in one volume of PBS/1mg/ml BSA. Mix well
- Wash them 2x with medium wihout any addition and resuspend them at 3x105
cells/ml in the same medium. To 6x106 cells in 20 ml of RPMI add 250l of 2M
glucose and 20l of 0.1M MnCl2. Mix by inverting the tube.
- In experiments where RGD peptides are tested cells are preincubated with the
respective reagent for 30min at 37 C prior addition to the wells
6) Plate cells in wells. 5ml/well (1.5x106 cells per well). Cells should be enough to cover
the well in only one layer). Spin plate at 200rpm 4min. Incubate at37C for 30-60 and 120
min.
7) Recover cells at indicated times. Wash gently once with PBS and lyze cells in RIPA
buffer at 4 C, or in SB1x (100l) and boil
8) Clarify cell lysate by centrifugation.
9) For IP, preclear with Prot-A-agarose for 1h 4C
Incubate with the antibody to precipitate coupled to Prot-A-Agarose beads for
2h at4C
Harvest immune complexes by quick centrifugation and wash them with RIPA
buffer
Boil them in 2xSB resolve them with 7.5% gels and transfer to nitrocellulose.
146
AUROPROBE INDIRECT IMMUNOLOCALIZATION

Solution I : PBS+5% BSA + 4% Goat serum
Solution II : PBS+1% BSA + 1% Goat serum
1) Recover sperm in PBS and wash once. Falcon tubes, 2.5ml max volume 1000 rpm x
10min.
2) Resuspend them in 500 l of PBS and count.
3) Adjust the concentration to 1 x 107 sperm/ml. At this point the sperm can be resuspend
in CM if capacitation is needed. Wash them with PBS afterwards.
4) Wash cells with solution II. 3 times with 500 l each (microfuge #3, 3 min)
5) Resuspend gently in solution I (107 cells/ 100l)
6) Separate sample in 4 (2 experimental, one positive and one negative control)
7) Add 25 l of antibody solution diluted in solution I at 100g/ml.
8) Incubate 30 min, RT, mix twice.
9) Wash sperm in solution II twice (#3, 3)
10) Resuspend pellet in 25 l of solution I. Add 25 l of diluted Auroprobe LM (antimouse IgM+ IgG conjugated with gold particles diluted 1/40 with solution I,
Amersham).
11) Incubate 60 min at RT and wash as before.
12) Resuspend cells in 200 l of solution II
13) Spin cells down in a cytospin centrifuge 5 min @ 750 rpm. Use 100 l per well to
have duplicates of each sample.
14) Allow cells to dry.
15) Fix them 30 sec in 45% Acetone, 9.25% HCOH in PBS (15 ml PBS, 22.5ml Acetone,
12.5ml 37% formaldehyde).
16) Rinse slides 3x3 in distilled water.
17) Remove excess of water and cover cells with silver enhancement mixture 6-18 min.
Watch samples to
check grain formation.
18) Wash 2x5 in distilled water. Dry and mount with Merckoglass. Leave at 4oC until
scoring.
147
COOMASSIEE BLUE STAINING OF MOUSE SPERM TO TEST ACROSOME

REACTION
3) Capacitate sperm in CM for 60 min at a concentration of 2-3 x 107 sperm/ml (37oC, in
5%CO2/air mix). Then, prepare tube samples indicated below and add sperm to a final
concentration of 3 x 106 sperm/ml (use polystirene tubes only).
4) After 20 min incubation take an aliquot of 10 l and place it in a tube containing 100 l
of 5.5% HCOH in PBS/0.4% PVP (polyvinyl pyrrolidone 40).
5) Incubate for 10 min.
6) Wash once with PBS (5 min max. speed in microfuge)
7) Dry cells onto a glass slide.
8) Stain them with 0.04% C. Blue G-250 in perchloric acid 3.5% (5 ml HClO4 in 100 ml
distilled water + 40 mg CB) for 2 min.
9) Rinse with PBS and mount with mounting media. Score intact sperm (blue acrosome)
vs. acrosome reacted sperm (destained acrosome).
RESULTS
Preparation of samples
%AR
#
CM(l) .......(l)
#Intact
#AR
[......](unit)
*n= number sperm counted

NOTE: Sodium selenite is highly toxic. Handle carefully.
148
n*
%Intact
EFFECT OF ASTROCYTES ON EL-4 CELLS (ANTI-PY BLOT)

1- Take 10 ml of EL-4 cells, put them down in a 15 ml tube. Spin the cells (2000 rpm, 10
min, RT) and resuspend them in 10 ml warm 0.1% FCS/DMEM, count them.
a)
Put 3 x 106 cells in a sterile eppendorf tube spin again and resuspend in
300 l of same medium containing 100g/ ml of anti-Thy-1 Ab (cold)
(107 cells/ml). Incubate at 4C for 30min.
b)
Meanwhile, wash 10ml of EL-4 cells and 10ml of EL-4-f cells as

indicated before. Count them
c)
Resuspend cells at 107 cells/ml in 0.1%FCS/DMEM. Put them in

eppendorf tubes.
d)
When incubation with anti-Thy-1 is finished. Incubate all cells at 37C

(bath) for 5 min.
2- During these 5 min, prepare astrocytes.
21-day-old astrocytes are growing in
10%FCS/DMEM.
3- Aspirate medium of all wells. Add warm PBS to 8 wells (10%FCS/DMEM/2mM
HEPES to rest of the wells). Briefly swirl the PBS around. Aspirate the PBS and add the
EL-4 cells as follows. Additions every 20 sec.
Well N
Add
1A and 1B__________________________________________
2A and 2B__________________________________________
3A and 3B__________________________________________
4A and 4B__________________________________________
TIMING
4- ______Incubate for 10 min at 37C (on a warm plate)
5- ______Change plate to ice. (prepare a box to cool down the 8 wells only, put styrofoam
in the other half) and add 100 l of 2x RIPA buffer (2ml 2x RIPA + 80 l Vn + 40 l
EDTA + 4 l BAL). Swirl solution to mix and pipette up and down.
6- ______Place cell extract in eppendorf tubes already labelled (at
7- ______Sonicate and leave at 4C for 15-30 min.
8- ______Spin extract at max speed and discard pellet.
149
4C).
9- ______Precipitate all supernatant (200 l, 1 x106) with
chloroform-methanol
technique (400l methanol+100l chloroform+ 200l water).

10- ______Resuspend pellet in 50l of sample buffer + reducing agent (5% mercaptoethanol) + Vn, boil, and load 25l (0.5 x106 )per lane on a gel.
150
IMMUNOFLUORESCENCE BY FACS ANALYSIS (IN PLATE ASSAY)

1) Count cells.............
2) Place 2 x105 cells per sample in each well (96 well plate conical bottom)
3) Spin cells 5 min. at 1200 rpm, RT. Discard solution by flicking the plate once.
4) Add first antibody* (have dilutions ready and cold), 50 l/well
5) Incubate >20 min at 4C.
6) Add 200l of cold PBS/2.5% FCS. Spin and discard supernatant as before
7) Resuspend the cells in the second antibody dilution (anti-Rat FITC 1/100 in PBS/2.5%
FCS), 50l/well
8) Incubate with second antibody for >20 min at 4C.
9) Wash as indicated in step 6
10) Resuspend in 200 l of PBS/2.5% FCS. Store at 4C until reading.
Cells
First Ab
Dilution of first Ab
1........................................................................................................................................
2........................................................................................................................................
3........................................................................................................................................
4........................................................................................................................................
5........................................................................................................................................
6........................................................................................................................................
7........................................................................................................................................
8........................................................................................................................................
9........................................................................................................................................
10......................................................................................................................................
11......................................................................................................................................
12......................................................................................................................................
151
IMMUNOFLUORESCENCE FOR CONFOCAL MICROSCOPY

7) For adherent cells grow them on round coverslips in a 24-well plate. They should not go
over 60% confluency.
8) Rinse them with PBS. Add 500l and remove carefully, 2x
9) Add 4% para-formaldehyde (prepared from powder)(b). Incubate for 10 min(c) at RT.
10) Wash with PBS twice. Block whith 5mM glycine for 15min and rinse again.
11) Treat samples with 0.2% Saponin in PBS, 10min ( stock Saponin is prepared at 1% in
PBS and stored at 4C for a month). This step could be omitted if the antigen is a cell
surface antigen. Other detergent as 0.1% TX-100 can also be used.
12) Wash with PBS 3x
13) Block the coverslip with a protein solution, eg. 2.5%FCS in PBS for 30 min. Dilute all
antibodies in blocking solution.
14) Incubate with first antibody for 60 min 37C in a moisture chamber, make a 30l drop
on a parafilm and invert the coverslip on it.
15) Wash with PBS 3x, make 500l drops and wash 3x for 5min each.
16) Incubate with second antibody for 60 min at 37C. Prepare it in blocking. If staining of
the actin cytoskeleton is desired then add 50ng/ml of phalloidin-rodhamine (stock
solution is at 50g/ml in ethanol stored at 20C, HIGHLY TOXIC). , Alternatively
counterstain with 0.005% Evans Blue in PBS for 1 min (optional).
17) Wash with PBS as in step 9, last one with distilledwater.
18) Mount with 1 mini-drop of fluorosave, put coverslip and sealed with nail varnish when
using long coverslips. Store at 4C. If using Mowiol, put a 5l drop, mount, and let
polimerize for 30 min at RT, then store at 4C. Mowiol: 6g glycerol, 2.4g MOWIOL(488 from Hoechst), 6ml H2O, 12ml 0.2M Tris pH8.5, 2.5% DABCO (1,4-diazobicyclo(2.2.2.)octane)
a) Preparation of coverslips
Place them in 70% ethanol for 10 min. Then, one by one, dip them in 100% ethanol
and flame them. Put them in a sterile 24 well plate. Leave them under UV light in the
laminar flow for 20min.
b) Preparation of 4% formaldehyde
Warm PBS at 65C in water bath. Place everything in ventilated hood. 4g of pformaldehyde (Fischer) are added to the warm PBS and let stir for over 1 hr. It does not
need pH adjustment, but check with strips that is around 7. Add more PBS to complete
volume to 100ml and aliquot solution in 4 ml aliquots. Store at -20C.
c) FITC antibodies dilute 1/100. Rhodamine, 1/200. In blocking solution.
152
19) Take 1 x105 cells to put down per well. Spin the cells (2000 rpm, 5 min, RT) and leave
them in a minimum volume (15 l/well of 1 cm diameter)
20) Put them down on slides for 10 min (slides treated with polylysine)(a). Do not let them
dry
21) Add 4% formaldehyde (prepared from powder)(b). Incubate for 10 min(c).
22) Wash with PBS twice. Immersing slides slowly and taking them away immediately.
23) Treat samples with 0.2% Saponin in PBS + 2.5%FCS, 10min ( stock Saponin is
prepared at 1% in PBS and stored at 4C for a month). This step could be omitted if the
antigen is a cell surface antigen. Other detergent as 0.1% TX-100 can also be used.
25) Incubate with first antibody for 15 min
27) Incubate with second antibody for 15 min. Prepare it in 0.2% Saponin solution (d)
29) Counterstain with 0.005% Evans Blue in PBS for 1 min
30) Wash 3-4x
31) Mount with 1 drop of fluorosave, put coverslip and sealed with nail varnish when using
long coverslips. Store at 4C.
a) Preparation of slides
Slides are 3 well slides. Dip them in acetone (coating has to be acetone resistant).
Then washed them in plenty of distilled water. Let them dry.
Polylysine from Sigma is diluted just before use 1/10 fold (final concentration is
0.01%).
Place 100l/well and incubate for 10min. Rinse them with water and they are ready
to use.
b) Preparation of 4% formaldehyde
Warm PBS at 65C in water bath. Place everything in ventilated hood. 4g of pformaldehyde (Fischer) are added to the warm PBS and let stir for over 1 hr. It does not
need pH adjustment, but check with strips that is around 7. Add more PBS to complete
volume to 100ml and aliquot solution in 4 ml aliquots. Store at -20C.
c) When using anti-Thy1 antibodies fixation needs to be longer than 1 hr otherwise it
induces crosslinking of thy-1 molecules (Science, 1994)
d) FITC antibodies dilute 1/100. Rhodamine, 1/200.
For 1ml solution add 10 l of FITC second antibody, 100l of 1% Saponin, 100l
FCS, and complete to 1 ml with 0.2% Saponin/FCS solution.
153
INDO-1/FACS
Measuring intracellular calcium
1) Count cells.............
2) Spin cells 10 min. at 2000 rpm, RT
3) Tap pellet to loosen it up and wash 1x with PBS-2%FCS, pH7.0 (P-2)
4) Tap pellet and resuspend at 2x106cells/ml of P-2.
5) Add 2M INDO-1 AM (0.4% final DMSO). Stock is at 500M, so add it at a final
dilution of 1/250.
6) Mix gently and immediatly. Put at 37C for 1 hr rotating.
7) Wash once with P-2 using a sharp spin and quickly resuspend the cells.
8) Resuspend in Calcium buffer (Ca-B) containing 137mM NaCl, 2.7mM KCl, 1mM
CaCl2, 1mM MgCl2, 1mM Na2HPO4, 25 mM glucose, 20mM Hepes, pH 7.4 and 1%
FCS. Final concentration should be 1x106 cells/ml
9) Keep cells at room temperature and in the dark until assay. Use cells before 2 hr after
loading. Cells are placed in 37C bath before assaying.
10) No cell activation is the zero of the assay and cells treated with 2M calcium ionophore
is the maximum value. Stock is at 500M so dilute 1/250.
11) Stimulus or ionophore are added right before assay by flow cytometry using FACS.
154
PY-IIF OF ASTROCYTES INCUBATED WITH EL-4 CELLS

1. One plate of 10cm with confluent astrocytes. Detach cells with a sterile rubber
policeman. Wash them 2x with complete medium (CM) and separate them (tap the
tube). Resuspend them in 7 ml of CM.
2. Prepare round coverslips: Place them in 70% ethanol for 10 min. Then, one by one, dip
them in 100% ethanol and flame them. Put them in a sterile 24 well plate. Total of 12
wells needed.
3. Add 500 l of astrocytes per well (12 wells with coverslips) and leave them growing for
24 h
Date...............
1) Prepare protein-A beads with expressed proteins. Incubate for 1 hr, 4C
2) When the hour incubation is about to finish, prepare astrocytes. Aspirate medium, add
warm PBS to wells, briefly swirl PBS around and discard. Add 200 l of PBS/2.5%
FCS. Immediately add the prepared beads.
A
B
C
D
4) Wash very gently with warm PBS. Aspirate add 600l of PBS/well. Repeat 3x
5) Fix cells with 4% HCOH during 10 min
6) Wash 2x with PBS containing 5mM glycine and 2x with PBS
7) Permeabilize with 0.1% Triton X-100 in PBS/1mMVanadate for 4min
8) Block with 0.5%gelatin in PBS/Vn for 10min at RT
9) Add 4G10 1/100 in blocking buffer only to 6 samples, do incubation over a parafilm.
Make 30l drops over parafilm and invert the coverslips over the drop. Incubate 1h at
37C
10) Wash with PBS/Vn 3x. Make drops of 500l and wash putting the coverslip in the drop
for 5min 5x.
155
11) Incubate 1h at 37C with anti-mouse-FITC diluted 1/200 in blocking buffer. Wash as
before. Last one with water and mount immediately with Mowiol: 6g glycerol, 2.4g
MOWIOL(4-88 from Hoechst), 6ml H2O, 12ml 0.2M Tris pH8.5, 2.5% DABCO (1,4diazobicyclo-(2.2.2.)octane)
12) Copy fields with confocal microscopy in optical disk.
156
PY-IIF OF ASTROCYTES INCUBATED WITH THY-1FC MOLECULES

1. One plate of 10cm with confluent astrocytes. Detach cells with a sterile rubber
policeman. Wash them 2x with complete medium (CM) and separate them (tap the
tube). Resuspend them in 14 ml of CM.
2. Add 500 l of astrocytes per well (24 wells with coverslips) and leave them growing for
48 h
Date...............
1) Prepare protein-A beads (50l) with expressed proteins (2g). Incubate for 1 hr, 4C.
Wash twice with cold PBS. Resuspend in 100l.
2) When the hour incubation is about to finish, prepare astrocytes. Aspirate medium, add
warm PBS to wells, briefly swirl PBS around and discard. Add 200 l of PBS.
Immediately add the prepared beads (50l/well, RT).
A
B
C
D
3) Incubate for ................. min at 37C
4) Wash very gently with cold PBS/5mM EDTA/1mM Vn. Aspirate add 1ml of PBS/well.
Repeat 2x
5) Resuspend cells in 100l 1xSB+DTT.
6) Put in an eppendorf tube and sonicate. Boil and load on a gel.
157
PY-IIF OF ASTROCYTES INCUBATED ON THY-1 COATED COVERSLIPS

-
Prepare round coverslips: Place them in 70% ethanol for 10 min. Then, one by one,
dip them in 100% ethanol and flame them. Put them in a clean plate. Total of 10
wells needed.
With the proteins to test, make 50l drops over parafilm and invert the coverslips over
the drop. Incubate o.n. at 4C.
PROTEIN
Wash once with PBS. Place in a 24-well plate
Block with 1% BSA in PBS. Leave 1 set without blocking. Add 200l/well and
incubate for at least 2 hrs at 37C.
Wash 4x with PBS
Prepare astrocytes. One plate of 10cm with growing astrocytes (non-confluent).
Aspirate medium. Rinse 2x with PBS without Calcium and Magnesium
Detach cells with trypsin or trypsin EDTA treatment. Add trypsin solution at 37C
and wait for 1-2 min.
Collect cells adding 3ml of PBS disggregating with a pipette and add one volume of
medium. Mix well
Wash them 2x with PBS and resuspend them at 1x106 cells/ml in PBS. Take 2x106
cells bring to 10ml with PBS, add 125l of 2M glucose and 10l 0.1M MnCl2.
Add 500 l of astrocytes per well (10 wells with coverslips) and leave them growing
for 3 h
Fix cells with 4% HCOH during 30 min
Wash 2x with PBS containing 5mM glycine and 2x with PBS
Permeabilize with 0.1% Triton X-100 in PBS/1mMVanadate for 4min
Block with 0.5% gelatin in PBS/Vn for 10min at RT
Add 4G10 1/100 in blocking buffer, do incubation over a parafilm. Make 30l drops
over parafilm and invert the coverslips over the drop. Incubate 1h at 37C
Wash with PBS/Vn 3x. Make drops of 500l and wash putting the coverslip in the
drop for 5min 5x.
158
Incubate 1h at 37C with anti-mouse-FITC diluted 1/200 in blocking buffer. Wash as

before. Last one with water and mount immediately with Mowiol: 6g glycerol, 2.4g
MOWIOL(4-88 from Hoechst), 6ml H2O, 12ml 0.2M Tris pH8.5, 2.5% DABCO
(1,4-diazobicyclo-(2.2.2.)octane)
Copy fields with confocal microscopy in optical disk
TEMPLATE FOR ASSAY

A
1
2
159
STAINING OF CELLS WITH CMFDA GREEN AND CMTMR ORANGE RED

(MOLECULAR PROBES)
1) Count cells..................
Dilute or concentratate them at 1 x 106 cells/ml in complete medium. Add dye
CMFDA:
5mM stock solution in DMSO

for staining 2.5M (10l in 20ml of CM for 2 x 107 cells)
CMTMR:
5mM stock solution in DMSO

for staining 4.5M (18l in 20 ml of CM for 2 x 107 cells)
2)Incubate cells with the dye for 30 min at 37C in the dark (incubator with CO2).
3) Wash once with medium and resuspend in same volume of CM. Incubate 60 min at 37C
(spontaneous release).
4) Wash twice in PBS/2.5% FCS and resuspend in same solution at 107 cells/ml.
5) While washing cells, prepare astrocytes. Aspirate medium, add warm PBS to wells,
briefly swirl PBS around and discard. Add 200 l of PBS/2.5% FCS. Immediately add
50l of EL-4 cells (50l of each type).
6) Spin plate 1-2 min 2000rpm
8) Wash very gently with warm PBS. Aspirate add 600l of PBS/well. Repeat 5x
9) Add 300l PBS/2.5% FCS
10)Copy fields with confocal microscopy in optical disk
TEMPLATE FOR ASSAY
A
B
C
D
160
RESULTS
Stimulating 2B4 cells on plates for anti-PY blots
A
B
C
D
Preparing the plates

1. Dilute antibodies in sterile PBS at 10 g/ml
Antibody
Dilution
Antibody
Dilution
2. Add 500 l/well to a 18mm well, flat bottom plate (petri dish). Can also use 5 ml falcon
tubes.
3. Incubate for 1hr, at 37C (CO2 incubator)
NOTE: the antibody solution could be recovered to be used again. Keep sterile.
4. Wash plates 3x with PBS (500 l/well each time)
NOTE: the plate could be stored at 4C, keeping wells with PBS and wrapping plate
with parafilm to avoid evaporation
DATE.........
5. Count cells..................
Wash them once in sterile PBS and resuspend them at 1 x 107 cells/ml in complete
medium with only 0.1% FCS.
6. Immediately before adding cells aspirate the PBS
7. Add 100 l of cell suspension, i.e. 1 x 106 cells per well
8. Incubate for different times at 37C (2, 4 and 10 min)
161
9. At every time point quickly stop reaction by adding 100l of cold 2x RIPA (2ml 2x
RIPA + 80 l Vanadate 50mM, 40 l 0.5M EDTA, 4l BAL:protease inhibitors) buffer
and recover cells pipetting up and down and placing them in an eppendorf tube.
10. Sonicate. Leave at 4C for 15-60 min
11. Spin 5min max speed and eliminate the pellet.
12. Supernatant is then precipitated with Chloroform-methanol technique
13. Dry samples, resuspend them in 50 l of sample buffer + reducing agent +Vn. Load on
a gel (25l/lane)
162
STIMULATING EL-4 CELLS ON PLATES FOR ANTI-PY BLOTS

Count cells..................
Wash them once in sterile PBS and resuspend them at 3.75 x 106 cells/ml in complete
medium with only 0.1% FCS.
2. Add 50 l/well to a 96-well, round bottom plate (Greinier)
3. Incubate for >6hr, at 37C (CO2 incubator)
NOTE: the antibody solution could be recovered to be used again
4. Wash plate 3x with PBS (200 l/well each time)
6. Add 40 l of cell suspension, i.e. 1 x 105 cells per well
7. Incubate for different times at 37C
8. At every time point stop rection by adding 40 l of 2x (sample buffer+ reducing agent +
vanadate)
9. Boil immediately
10. Load on a gel
163
STIMULATING IL-2 PRODUCTION ON EL-4 CELLS

Count cells..................
ALL MATERIAL AND SOLUTIONS NEED TO BE STERILE

1. Spin cell suspension in eppendorf tubes 5min at 2000 rpm, RT. Discard supernatant.
2. 5x 106 cells/ml are exposed to the first antibody at saturating concentrations and
incubated for 45 min, 4C
3. Cells are washed once with cold PBS containing 2% FCS.
4. Add to cells the second antibody to crosslink the first one. Second antibody is used at
50 g/ml
5. Incubate for 45 min, 4C
6. Wash once as before (step 3)
7. Resuspend in complete medium (800l) with or without 10ng/ml of phorbol ester
8. Plate in cuadruplicate at 105/well (200l/well)
9. Incubate for 48 hr at 37C
TUBE #
FIRST ANTIBODY
SECOND ANTIBODY
1
2
3
4
5
6
7
8
9
10
11
12
SECOND PART
Measuring IL-2
1. Spin down the plate for 10 min, 2000rpm, RT
2. Harvest supernatants: take 150l/well and transfer them to another plate to use them in
the IL-2 assay. KEEP FORMAT OF THE TEMPLATE BELOW. Test supernatants in
serial dilutions and save supernatants left at -20C.
To prepare plates add 100l of CM/well to 96well flat bottom plates. Add then,
100l of each supernatant to first column and dilute towards the right of each row
(12 dilutions). IL-2 standard curve starts at approx. 150 U/ml.
164
3. Count CTLL cells (...............) and wash them three times with complete medium
without IL-2 (10% FCS is the CM that Marga gives me)
4. Resuspend cells in complete medium (no IL-2) at 5x104/ml
5. Plate them at 5x103 cells per well in aliquots of 100 l
6. Incubate for 22hr at 37C
7. Add 0.5Ci of 3H-thimidine/well and incubate per 6hrs (dilution 1/10 in CM)
8. Process samples for counting in a septrometer.
165
STIMULATING EL-4 CELLS TO PRODUCE IL-2 ON PLATES (OTHER)

Count cells..................
Wash them once and resuspend them at 106 cells/ml
ALL MATERIAL AND SOLUTIONS NEED TO BE STERILE
2. Add 50 l/well to a 96-well, round bottom plate (Greinier)
3. Incubate for >6hr, at 37C (CO2 incubator)
NOTE: the antibody solution could be recovered to be used again
4. Wash plate 3x with PBS (200 l/well each time)
DATE..........................
6. Add 100 l of cell suspension at 106 cells/ml
7. Incubate for 42 hr, at 37C (CO2 incubator)
SECOND PART
Measuring IL-2 production
1. Spin down the plate for 10 min, 2000 rpm, RT
2. Harvest supernatants and transfer them to another plate to use them in the IL-2 assay.
Test supernatants in serial dilutions and save supernatants left, at -20C
3. Prepare dilutions of supernatants to test. Need also a standard curve with IL-2 serial
dilutions starting from 80 U/ml. Place 100l of complete medium per well. Add 100l
of supernatant in the first column and do serial dilutions towards the right of the plate.
4. Count CTLL cells (...............) and wash them twice with medium without IL-2
5. Resuspend cells in complete medium (no IL-2) at 5x104/ml
6. Plate them on the diluted supernatants at 5x103 cells per well per 100 l
7. Incubate for 22hr at 37C
8. Add 0.5Ci of 3H-thimidine/well and incubate per 6hrs
9. Process samples for counting in a septrometer
166
TESTING ACROSOME REACTION IN HUMAN SPERM

1. Prepare cells via Percoll gradient
sperm sample (1ml)

47% Percoll
(2ml)
90% Percoll
(2ml)
2. Spin down at 1800 rpm (Beckmann Clinical centrifuge)

3. Recover sperm pellet and wash it with TNC buffer once.
centrifugation at 3500 rpm, Beckmann clinical centrifuge.
Washes are done by
Resuspend pellet in 1ml of
TNC buffer and count sperm number.

4. Capacitate sperm in F-10 medium at a concentration of 1 x 107 sperm/ml (37oC, in
5%CO2/air mix) for 3 hrs. Then, prepare tube samples indicated below and add sperm
to a final concentration of 3 x 106 sperm/ml (use polystirene tubes only).
Coommasie Blue staining technique
1. After 30 min and 3 hrs incubations take an aliquot of 10 l and place it in a tube
containing 100 l of 5.5% HCOOH in PBS/0.4% PVP (polyvinyl pyrrolidone 40).
2. Incubate for 10 min.
3. Wash once with PBS (5 min max. speed in microfuge)
4. Dry cells onto a glass slide.
5. Dip slide in PBS. Stain sperm with 0.04% C. Blue G-250 in perchloric acid 3.5% (5 ml
HClO4 in 100 ml distilled water + 40 mg CB) for 2 min.
6. Rinse with PBS and mount with mounting media. Score intact sperm (blue acrosome)
vs. acrosome reacted sperm (destained acrosome).
Lectin (PSA) staining technique
167
1. After 30 min and 3 hrs incubations remove 10 l aliquot into 500 l of TNC and spin
in microfuge #4.
2. Resuspend the pellet in 95% ethanol. Incubate for at least 30 min. Dry sperm onto
microscope slide.
3. Cover the sperm with 50 l of rodhamine-labelled PSA (100 g/ml in PBS) and
incubate 10 min at room temperature. (dark)
4. Rinse away the unbound PSA by dipping the slide into a beaker of deionized water and
moving it gently back and forth for about 15 sec.
5. Mount with gelvatol.
168
TREVORS FIXATIVE
(good for cytoskeletal preparations)
20g p-formaldehyde (100%) in 400ml of dd water and heat to 60C
Add 2ml of 10M KOH (it should solubilize)
Cool down
Add 100ml of (0.5M PIPES and 10nmM EGTA and 10mM MgCl2, pH to 6.8 at RT)
Solution has approx. 500ml total with 100mM PIPES, 2mM EGTA, 2mM MgCl2
Filter to eliminate unsolubilized material
After fixing 15min with this solution, wash 3x with 150mM Tris-HCl at pH 7.4-7.5
(IMPORTANT!!)
169
(ALPHA-32P)GTP BLOT ANALYSIS

4. Prepare samples of activated vs nonactivated cells
5. Run them on a gel (12.5%, rather small Mr proteins are expected)
6. Incubate gel in transfer buffer 30 min before transfering (Tris/glycine, 20% methanol,
0.05% SDS)
7. Transfer to nitrocellulose 0.2m (1 hr 100V)
8. Incubate membrane in 20mM Tris pH7.5, 1mM MgCl2, 0.3% Tween 20, 2x 10 min at
room temperature.
9. Incubate blot for 30 min in the same buffer containing 0.3% BSA and alpha-32P-GTP
(3l of 3000Ci/mmol in 10ml, final is 1Ci/ml)
10. Wash 2x in same buffer without BSA for 15 min.
11. Dry nitrocellulose, wrap it with Saran wrap and expose to film (sometimes 30 min
exposure is sufficient)
12. After exposure, nitrocellulose can be blocked and use for immunoblot (using ECL to
develop)
170
ANTI-THY-1 BLOTS
Washing Buffer (WB), 1 lt
Tris 50mM/NaCl 0.15N or PBS 1x
1 lt
Tween-20
2 ml
1) Block membrane for 1 hr in WB + 3.5% Milk (fat-free) and sodium azide 10nM
2) Rinse membrane with WB twice
3) Incubate with anti-Thy-1 pAb (1/3.000 in WB) for 1 hr.
4) Wash membrane with WB 5x7 min each.
5) Incubate with anti-rabbit-HRP (1/3.000 in WB) for 1 hr.
6) Wash as indicated in step #3.
7) Detect the enzyme with chemiluminescence substrate (ECL, Amersham International).
8) Expose the membrane to an X-ray film for the time needed (3 sec. to 30 min).
9) If too much background is a problem wash the membrane again twice (15 min each)
with WB containing 10 nM NaN3, then repeat steps 6 and 7.
Notes:
-If using PVDF membrane, Immobolon should never dry, if so reincubate with pure
methanol for 30 sec. and equilibrate with transfer buffer then WB before continuing with
the blot.
-Use different boxes for each incubation.
-Do washes in boxes where the membrane can move freely and use a lot of WB.
-After developing with ECL do not leave the membrane to dry and save it at 4C.
171
EFFECT OF ASTROCYTES ON EL-4 CELLS (ANTI-PY BLOT)

1) Take 10 ml of EL-4 cells, put them down in a 15 ml tube. Spin the cells (2000 rpm, 10
min, RT) and resuspend them in 10 ml warm 0.1% FCS/DMEM, count them.
Put 3 x 106 cells in a sterile eppendorf tube spin again and resuspend in 300 l of same
medium containing 100g/ ml of anti-Thy-1 Ab (cold) (107 cells/ml). Incubate at 4C for
30min.
Meanwhile, wash 10ml of EL-4 cells and 10ml of EL-4-f cells as indicated before. Count
them.
Resuspend cells at 107 cells/ml in 0.1%FCS/DMEM. Put them in eppendorf tubes
When incubation with anti-Thy-1 is finished. Incubate all cells at 37C (bath) for 5 min.
2) During these 5 min, prepare astrocytes.
10%FCS/DMEM.
21-day-old astrocytes are growing in
Aspirate medium of all wells. Add warm PBS to 8 wells (10%FCS/DMEM/2mM HEPES
to rest of the wells). Briefly swirl the PBS around. Aspirate the PBS and add the EL-4 cells
as follows. Additions every 20 sec.
Well N
Add
1A and 1B__________________________________________
2A and 2B__________________________________________
3A and 3B__________________________________________
4A and 4B__________________________________________
TIMING
______
3) Incubate for 10 min at 37C (on a warm plate)
______
4) Change plate to ice. (prepare a box to cool down the 8 wells only, put
styrofoam in the other half) and add 100 l of 2x RIPA buffer (2ml 2x RIPA
+ 80 l Vn + 40 l EDTA + 4 l BAL). Swirl solution to mix and pipette up
and down.
______
5) Place cell extract in eppendorf tubes already labelled (at
______
6) Sonicate and leave at 4C for 15-30 min.
______
7) Spin extract at max speed and discard pellet.
______
8) Precipitate all supernatant (200 l, 1 x106) with chloroform/methanol

technique (400l methanol+100l chloroform+ 200l water).
______
9) Resuspend pellet in 50l of sample buffer + reducing agent (5% mercaptoethanol) + Vn, boil, and load 25l (0.5 x106 )per lane on a gel.
PREPARE BEFORE STARTING

1) 2x 10% gels
172
4C).
2) DMEM with 10% FCS, warm up

3) DMEM with 0.1% FCS, warm up
4) DMEM with 10% FCS and 2mM HEPES, warm up
5) warm block
6) warm bath
7) PBS sterile, warm up
8) ice box
9) labelled eppendorf tubes (1-4, A and B)
173
EXPRESSED PROTEINS PURIFICATION FROM HEK-293 SUPERNATANTS

Washing buffer: PBS 1x
Elution buffer: citric acid pH 2.5
1) Equilibrate the column (high trap, protein-A Sepharose, Pharmacia) with 50 ml of PBS
2) Load the column with 400 ml supernatant which has been previously cetrifugated and
filtered (0.45m). Add NaN3. The supernatant is passed over and over the column during
the night at 4C. Recover the flow through.
3) Wash the column with 15ml of cold PBS using a syringe as a pression device
4) Prepare tubes for elution: Add 200l of 1M Tris pH 7.4 per tube and labelled them
5) Elute the column with Elution buffer and collect fractions of 1 ml. 8 fractions is
enough.
6) Mix the eluate with the Tris after every tube collection
7) Monitor for protein at OD=280nm and pool those fraction containing the antibody
The column should be washed immediately with 80ml of PBS. If the column is not needed
the same day wash with 20ml of PBS containing azide.
Repeat steps 2-7 until with 400ml of fresh supernatant everytime
8)Concentrate and dialyze with sterile PBS the antibody using Centricon 30 (Amicon)
(Tube #)A 280nm
1-------------5-------------2-------------6-------------3-------------7-------------4-------------8--------------
174
LIGAND BLOT ANALYSIS

9. Samples are boiled under non-reducing conditions for 7 min
10. Spin down non solubilized material
11. Add reducing agent and boil again for 3 min
12. Run gel. PAGE-SDS
13. Incubate gel in transfer buffer for 5 min
14. Transfer gel to 0.2 m nitrocellulose, 35V o.n. or 2hrs, 80V
15. Stain with Ponceau red; destained well with water
16. Incubate NC with 3% NP-40 in PBS for 30 min, RT (PBS can be replaced by TN or
TNC in all following steps.
17. Incubate then with 1% BSA in PBS for 3 hr, RT
18. Then 10 min with 0.1% Tween 20 in PBS, RT
19. Incubate in a solution with 300,000cpm/ml of 125I- LL95/ PBS/1%BSA/0.1% Tween
20. The volume of ligand solution used is important. I normally incubated a strip of
NC in a 15 ml Falcon tube filled with approx. 12-14 ml. The solution has to be cold
and the incubation is at 4C, o.n. shaking.
20. Prepare 0.1% Tween 20 in PBS and PBS to have cold for the washes of the following
day.
21. Washes I do changing the strip of NC to a new Falcon tube and I filled them with 14 ml
of washing solution/wash. 5 washes, 10 min each with Tween and then 2 with PBS.
All at 4C and shaking.
22. Dry the NC and expose to X-ray film.
175
PY BLOTS
Washing Buffer (WB), 4 liters.

NaCl
35 g
Tris-OH, pH 7.6
24 g
Tween-20
2 ml
Gelatin (From cold water fish skin, Sigma G-7765) 8 ml

Thimerosal
4g
1) Block Immobilon for 1 hr in WB + 1% gelatin

2) Incubate with PY20 (1 :g/ml) for 1 hr.
3) Wash with WB 5x7 min each.
4) Incubate with anti-mouse-HRP (0.25 Fg/ml) for 1 hr.
5) Wash as indicated in #3.
6) Detect the enzyme with chemiluminescence substrate (ECL, Amersham International).
After incubating with the substrate for 1min, remove excess of liquid and wrap the
membrane with plastic-wrap.
7) Expose the Immobilon to an X-ray film for the time needed (3 sec. to 30 min).
8) If too much background is a problem wash the Immobilon again twice (15 min each)
Notes:
-Immobolon should never dry, if so reincubate with pure methanol for 30 sec. and
equilibrate with transfer buffer then WB before continuing with the blot.
-Use different boxes for each incubation.
-After developing with ECL do not leave the membrane to dry.
176
SPOT BLOT
Washing Buffer (WB): Tris 50mM/NaCl 0.15N or PBS 1x 1 lt
Tween-20
2 ml
10) Dry down the Ab to test. 2g/circle in the nitrocellulose piece

11) Block membrane for 1 hr in WB + 2% BSA
12) Rinse membrane with WB
13) Incubate the nitrocellulose with a solution containing the Ag (15g/5ml of WB 0.1x)
14) Wash membrane with WB 5x7 min each
15) Wash membrane with PBS twice
16) Cut circles with proteins and solubilize in SB1x
17) Run on an 8% gel and transfer to nitrocellulose to do a Western blot
18) Incubate with anti-Thy-1 pAb (1/3.000 in WB) for 1 hr
19) Wash membrane with WB 5x7 min each.
20) Incubate with anti-rabbit-HRP (1/3.000 in WB) for 1 hr.
21) Wash as indicated in step #10.
22) Detect the enzyme with chemiluminescence substrate
23) Expose the membrane to an X-ray film for the time needed (3 sec. to 30 min).
24) If too much background is a problem wash the membrane again twice (15 min each)
Notes:
IfusingPVDFmembrane,Immobolonshouldneverdry,ifsoreincubatewithpuremethanolfor
30sec.andequilibratewithtransferbufferthenWBbeforecontinuingwiththeblot.
- Use different boxes for each incubation.

-After developing do not leave the membrane to dry, wrap it with a plastic wrap and save it
at 4C.
177
STIMULATION OF EL-4 CELLS (ANTI-PY BLOT)

1) Take 12 x 106 of EL-4 cells (for 12 different points). Spin the cells (2000 rpm, 5 min,
RT) and resuspend them in warm 10% FCS/RPMI/5mM -mercaptoethanol/antibiotics
(CM), at 107 cells/ml.
2)Place 1x106 cells (100l) per eppendorf tube, spin down at 2000rpm for 4 min and add
the following:
Tube#
Add
1__________________________________________
2__________________________________________
3__________________________________________
4__________________________________________
5__________________________________________
6__________________________________________
7__________________________________________
8__________________________________________
9__________________________________________
10__________________________________________
TIMING
______
______
4) Spin max speed few seconds ( ). Discard supernatant.
______
5) Add second antibody warm.
Tube#
Add
1__________________________________________
2__________________________________________
3__________________________________________
4__________________________________________
5__________________________________________
6__________________________________________
7__________________________________________
8__________________________________________
9__________________________________________
10__________________________________________
______6) Incubate for 5 min at 37C.
______7) Add 800l of cold PBS/5mM EDTA/1mM Vn quickly mix and spin at max speed
for 15 sec.
______8) Resuspend in 100l of RIPA buffer(2ml 1x RIPA + 80 l Vanadate 50mM, 40 l
0.5M EDTA, 4l BAL:100g/ml benzamidine/ 10g/ml Antipain/2.5g/ml Leupeptin)
______9) Sonicate. Leave at 4C for 15-60 min
_____10) Spin 5min max speed and eliminate the pellet.
_____11) Supernatant is then precipitated with Chloroform-methanol technique: 100 l, 1
x106 + 400l methanol+100l
chloroform+ 300l water.
_____12) Dry samples, resuspend them in 50 l of sample buffer + reducing agent +Vn.
Load on a gel (25l,0.5x106 cells/lane).
178
IMMUNOPRECIPITATION
1) Prewashed Protein-A beads twice with PBS 1x. 50 l of beads per tube.
2)Take ..........l of thawed extract and add them to the eppendorf tubes containing the protA beads
3) Incubate for 2hr at 4C on the rotating wheel.
4) Spin beads 15 sec, max speed
5) Wash the beads with cold extract buffer(.............) ......x. Then wash 2x with cold PBS
6) Solubilize proteins from the beads with sample buffer + DTT(.........l/tube). Boil for 3
min spin, mix, spin and load.
7) Run on a .........% gel. Load ........l per lane.
179
IMMUNOPRECIPITATION WITH LL95

RIPA extract of any cell will be maintained at room temperature
immunoprecipitation procedure, so add proteinase inhibitors (PIC I and Pic II).
during
1) Pre-adsorb the extracts with 30-50 l of Rat anti-mouse kappa chain-Sepharose beads
for 15 min.
2) Spin down beads in microfuge (set it at #4 and spin for 3 min). Recover supernatant in
a fresh eppendorf tube.
3) Add 20 g of LL95 and incubate, shaking for 30 min.
4) Add 100 l of fresh beads and incubate as before for 2 hrs.
5) Spin the beads down as before and then wash them with triton X-100 buffer (50 mM
tris-OH, 0.6 M NaCl, 0.5 % Triton X-100, pH 7.8), 4 times.
6) The beads now are ready to use in kinase assay, to elute with acid or SDS-sample buffer.
20-30 l of beads should contain enough protein to visualize it in a blot with PY20. For a
kinase assay wash the beads first with the kinase assay buffer without ATP.
180
F: CELL CULTURE
RAT ASTROCYTE CELL LINE DITNC...........................................................................182

THAWING SERUM...........................................................................................................183
HEAT INACTIVATION......................................................................................................183
CELL CULTURE, STARVATION AND STIMULATION OF SWISS 3T3 CELLS.........184
PROTOCOL FOR NUDE MICE EXPERIMENTS (FROM ERNST REICHMANN)......186
SF9 CELL CULTURE........................................................................................................187
DIFERENCIATION OF CAD CELLS. MORPHOLOGICAL TEST................................188
DIFERENCIATION OF PC12 CELLS- MORPHOLOGICAL STUDY...........................188
DIFERENCIATION OF PC12 CELLS FOR IMMUNOFLUORESCENCE.....................188
DIFERENCIATION OF CAD CELLS...............................................................................189
DIFERENCIATION OF PC12 CELLS...............................................................................190
TREATMENT OF EL-4-F CELLS WITH G418................................................................191
METHOTREXATE PREPARATION.................................................................................193
THAWING THE CELLS....................................................................................................194
FREEZING CELLS............................................................................................................195
ADAPTATION OF CELLS TO SERUM FREE MEDIUM (SFM)...................................196
HOECHST STAINING.......................................................................................................197
PREPARATION OF MONONUCLEAR CELLS FROM HUMAN PERIPHERAL BLOOD
.............................................................................................................................................198
CELL VIABILITY ASSAY (MTS).....................................................................................199
USE OF A HEMACYTOMETER......................................................................................200
MTT METHOD FOR THE EVALUATION OF MIGRATORY CELLS...........................201
MCF-7 CELLS MIGRATION ASSAY...............................................................................202
GROTH IN SOFT AGAR...................................................................................................203
181
RAT ASTROCYTE CELL LINE DITNC

Grow them in DMEM containing 10% FCS
To pass them scrape the plate with rubber policeman (or the rubber part of a sterile
syringe), recover them in a tube and spin them at 2000 rpm for 10 min.
Gently resuspend them in 1 ml of medium (this is the volume per 1x10mm plate which is
confluent)
Dilutions depend when the cells are needed to be confluent:
100 l/ 10 ml Confluent in approx.
4 days
200l
3 days
300l
2 days
To freeze them take 1x10mm plate/vial of cells to freeze.

resuspend in DMEM/10%FCS/5%DMSO. Freeze slowly.
182
Spin them as before and
THAWING SERUM
Remove serum from 20 C storage and place 24 h in a refrigerator at 2 to 6 C. Transfer
the bottles to a 37 C water bath, agitating periodically to mix the solutes concentrated at
the bottom of the container. Do not hold the serum at 37 C any longer than neccesary after
thawing. Thawing serum in a bath above 40 C without mixing may cook the
concentrated proteins in the bottom of the container and precipitates may form around the
inside of the bottle. Thawing serum at higher temperatures is not recommended.
Turbidity and flocculent material may be present after thawing or after prolonged freezer
storage. Our experience indicates these changes will not affect performance of the product.
The manufacturer (HyClone ) recommended the product be left in the photoprotective
yellow bag until used.
HEAT INACTIVATION
Thaw serum as recommended
Fill an equivalent bottle with water and place a thermometer in the water.
Fill a water bath above the serum line in the bottle .
Preheat the water bath to slightly higher than 56 C to allow for cooling when serum is
added.
Place the serum and the water in the water bath.
Mix the serum approximately every 5 minutes to a avoid gelling of the serum proteins.
When the temperature of the water, and consequently the serum, reaches 56 C beging
timing.
Allow the serum to heat for 30 minutes.
Continue mixing the serum every 5 minutes.
Whenthe30minuteheatinactivationperiodiscompleted,removetheserumfromthewaterbath
andrapidlyandsterily,putthealiquotsinsterilestubesandfreezeat20C.
183
CELL CULTURE, STARVATION AND STIMULATION OF SWISS 3T3 CELLS

Work under the hood. Use sterile PBS, medium, pipette, etc. Wash your hands with ethanol.
Solutions:
Medium: DMEM low glucose (1 g/l), (Fluka, 21 885-025)
with GlutaMAX and pyruvate without HEPES, without antibiotics. Stored at 4C.
PSN 100x: (GIBCO; 15640-048)
5 mg penicillin, 5 mg streptomicin, 10 mg neomycin/10 ml. Aliquots of 5 ml stored at
-20C.
FCS: (Sera Tech.; SO115/523H)
Aliquoted in 50 ml sterile tubes and stored at -20C. Once thawed, store it at 4C.
Trypsin solution (GIBCO; 25090-028)
2.5% (1:250) 10x.
Medium: 10% FCS ( fetal calf serum) in DMEM (low glucose medium). Prepare the
DMEM by first adding the antibiotics,immediately after opening the bottle, PSN ( 5 ml into
a bottle of 400 ml DMEM)
Cells are grown on sterile 10 cm dishes (Nunc, 150350) in 10 ml complete medium. They
are normally plated at 6x105 cells and require 3 days to reach 60% confluency,at which
point they should be passaged.
To passage cells:
- Remove the old medium with a sterile pipette
- Wash the cells with PBS. Add the buffer carefully down the side of the dish.
Remove PBS immediately and add 1 ml trypsin (dilute the stock 1:50 in PBS). All cells
should be covered with liquid. Incubate for 5 min at 37C.
- Detach the cells using a 1 ml pipetteman with blue tips by pipetting up and down slowly.
Avoid creating bubbles or foam.
- Put the cells into sterile 15 ml tube and fill the tube with sterile PBS or medium without
FCS.
- Centrifuge for 5 min at 1500 rpm, RT.
- Discard the supernatant by inverting the tube.
- Resuspend the cells in 1 ml medium with blue tips and transfer x ml (depending on cell
confluency required) into a new Nunc dish containing 10 ml medium.
STARVATION
Remove medium containing FCS (10%). Wash twice with PBS at RT.
Add medium containing 0.5% FCS. Incubate the cells for 20 hours at 37C.
Control:
No colocalization or low level of colocalization between PKC and caveolin-1.
184
Loss of actin stress fibers (phalloidin staining).

Cells are not apoptotic.
Less than 20% dead cells by Trypan Blue exclusion.
STIMULATION WITH EGF
Add EGF in DMEM/0.5% FCS to obtain a final concentration of 20 ng/ml.
Add 5 ml of medium containing EGF in a 10 cm Nunc dish. Incubate at 37C for required
time period.
Stop the stimulation by removing the medium. Wash briefly twice with ice-cold PBS if you
want to continue by doing a subcellular fractionation.
For immunofluorescence add paraformaldehyde fixative.
EGF: Epidermal Growth Factor (Boehringer; 855 731).
STIMULATION WITH TPA
TPA is diluted in DMSO (Dimethylsulfoxide, Fluka, 41640). Add TPA in medium
containing 0.5% FCS. 5 ml of medium is enough to stimulate cells of one 10 cm dish.
Incubate at 37C for required time period.
Stop the stimulation by removing the medium. Wash briefly with ice-cold PBS if you want
to continue by doing a subcellular fractionation.
For immunofluorescence add paraformaldehyde fixative.
Note: Phorbol ester is highly toxic. All phorbol ester waste must be treated in 2M NaOH for
24 hours before discarding.
185
PROTOCOL FOR NUDE MICE EXPERIMENTS (FROM ERNST REICHMANN)

Anaestesie: Diazepam (5 mg/kg ip) + Fentanyl (0,3 ml/kg ip. Gegenwrtig nehmen wir
KETALAR (100mg/ml) und RAMPUN 2%ig in 0,9% NaCl. Das Ganze wird erst kurz vor
der Injektion gemischt 1,2 ml KATALAR + 0,8 ml RAMPUN. Niemals nachspritzen, dann
sterben die Muse! Zum Nachbetuben nimmt man METAFONE auf Watte in einem 50 ml
Falcon Tube.
Protokol: Man injiziert dann je nach Fragestellung 100 000 - 1 000 000 Zellen in Medium
ohne Serum, subkutan. Auch die Zellen sollten 1x in Medium ohne Serum gewaschen dein.
Die Markierung der Muse kann durch kleine Zeichen im Ohr, oder durch Entfernen von
Zehen oder Fingern in einer bestimmten Reihenfolge geschehen. Die Kontrolle des
Tumorwachstums sollte ber 2-3 Monate erfolgen, bis zu 2x wchentlich (hierbei kann man
die Ausdehnung des Tumors in 2
Dimensionen mit einer Schieblehre messen - es gibt Computerprogramme, die das Volumen
errechnen. Haben die Tumoren eine Grsse von mehr als einem Quadratzentimeter erreicht,
sollten die entsprechenden Tiere abgesetzt werden.
English paper version of protocol (see Bener et al., 2000)
Tumorigenicity assays. 106 cells were suspended in 50 l DMEM and injected
subcutaneously into 6-8 week old nude mice. For each mouse, control cells (parental HT29
or DLD1 cells or mock transfected cells) were injected on the left and HT29 or DLD1 cells
transfected with caveolin-1 (clones C13, C14, C16 or C2, C4 respectively) on the right.
Large (D) and small (d) diameters of growing tumors were measured twice a week and
corresponding volumes (V) were estimated using the equation V=d2xDx/6. To re-isolate
tumor cells for further culture, the tumor tissue was excised, cut into small pieces under
sterile conditions using scalpel blades and digested with trypsin/EDTA for 15 min at 37C.
Tumor cells were cultured until confluent in 10 cm Petri dishes, trypsinized, diluted 1:10 in
fresh medium and seeded again. After a second passage, when tissue debris and
contaminating cells had been eliminated, ex-tumor cells were lyzed at 80% confluency and
processed for caveolin-1 detection as described.
186
SF9 CELL CULTURE

Media: Grace's Insect Media, Supplemented 1X liquid (with 3.3 g/l lactablumin
hydrolysate, with 3.3g/l yestolate, with L-glutamine without insect hemolymph), Gibco
350-1605AJ
Serum Supplement: To the above media add 10% Fetal Bovine Serum, heat-treated
(Gibco 230-6140PJ)
Antibiotics: Do not add antibiotics to the maintenance cultures, but to cultures which are
going to be used for baculovirus expression add pennicillin/streptomycin/amphotericin
(100X solution, Gibco 600-5240AG). Gentamycin may also be used in addition (Gibco )
Flask Culture: Sf9 cells grow well in tissue culture flasks if they are fed every other day
and passed at about 1:4 every 4 days. To pass the cells: Remove the media. Add fresh
media that has been warmed to 25 C. Using a pipet, direct a stream of media very gently
across the surface of the plate to detach the cells. Repeat the wash 3-4 times until most of
the cells have floated off the flask. Be careful not to cause foaming of the media. Transfer
1/4 to 1/5 of the cell suspension to a fresh flask and add fresh media. For a T-25 flask use
5 ml media, for T-75 use 12-15 ml. It is not usually necessary to culture with antibiotics if
cultures are grown in T-flasks.
Spinner flask culture: Sf9 cells grow well in spinner culture only if they are maintained
fastidiously. Spinner flasks must be immaculate before cells will grow in them. Our
procedure for cleaning the flasks is to autoclave them filled with water for 45 min, soak for
several hours to overnight in 1% NaOH, rinse with 0.1% HNO 3, rinse 10X with deionized
water, autoclave with deionized water for 45 min, drain and bake in 250 F oven overnight,
autoclave dry for 45 min plus 15 min dry cycle.
Freezing cells: Freeze in growth media plus 10% DMSO, freeze slowly (pack cells in a
styrofoam container and keep at -80 C overnight, then transfer to liquid N 2 storage), thaw
quickly. 4x106 cells per aliquot is a good number to store.
Note: Sf9 cells are extremely temperature sensitive and cannot tolerate temperatures over
30 C at all. Be careful of heating media in a 37 C bath to warm up, it is very easy to
overheat the media and kill the cells almost instantly.
187
DIFERENCIATION OF CAD CELLS. MORPHOLOGICAL TEST

3. Grow cells CADcells in DMEM-F12 medium with Pen/Strep, 8% FCS. Stock of
DMEM-F12 (Gibco-BRL #12400-024) is prepared in nanopure water. Add 1.2g per liter
of sodium bicarbonate, tissue culture grade and Pen/strp and store sterile filtered at 4 C.
4. Obtain cells in 10cm plate.
5. Recover cells from a well and resuspend them in 5ml of medium. Pass them through a
sterile needle (syringe) to separate cells and count them.
6. Plate 500.000 cells in a 10cm plate in medium. Let them adhere for to the plate
overnight (or more than an hour).
7. Chsange media to differentiation media, i.e., the same but without serum and
containing 50 ng/ml of sodium selenite (selenite stock is prepared at 10g/ml in sterile
medium without FBS, aliquoted in 30 l aliquots and stored frozen at 20C) to induce
differentiation
8. Follow the morphology of the cells by microscopy and draw the changes you observe
day by day.
9. Maintain cells for 2 weeks.
DIFERENCIATION OF PC12 CELLS- MORPHOLOGICAL STUDY
10. Grow cells PC12 in RPMI with Pen/Strep, 10% FCS.
11. Obtain cells in 10cm plate.
12. Recover cells and resuspend them in 5ml of medium. Pass them through a sterile needle
(syringe) to separate cells and count them.
13. Plate 500.000 cells in a 10cm plate in medium with 10% serum and let the cells adhere.
14. Once adhered change the medium for medium with 5% serum in the presence of
100ng/ml of NGF. (NGF stock is prepared at 20g/ml in sterile medium containing
10% FBS, aliquoted in 150 l aliquots and stored frozen at 20C) to induce
differentiation.
15. Follow the morphology of the cells by microscopy and draw the changes you observe
day by day.
16. Maintain cells for 2 weeks.
DIFERENCIATION OF PC12 CELLS FOR IMMUNOFLUORESCENCE
18. Obtain confluent cells in 6-well plate to keep the stock of cells.
19. Obtain confluent cells in a 10cm plate
20. Prepare coverslips in a 6-well plate
20.1.
Clean round coverslips (10-15mm) in 70% ethanol (at least 30 min)
20.2.
Pass them for 100% ethanol and flame them
20.3.
Place them in a sterile 6-well plate (3 per well)
20.4.
Prepare polylysine sterile at 0.01% (sterile filtered with a 0.2m filter with a
syringe)
20.5.
Add polylysine on the coverlips only. Leave for 10 min. Aspirate and
recover the polylysine in sterile condition and keep at 4C.
20.6.
Wash the coverslips 3x with sterile nanopure water
20.7.
Wash them again 3x with sterile PBS.
21. Add medium (2.5ml/well). Recover cells from 2 wells (see step 2) by trypsinization
(aprox. 5min). Wash with medium and resuspend in total medium.
22. Add 10.000 cells per coverslip and let the cells adhere
23. Then change medium with one containing 5% serum and NGF to induce differentiation
(100ng/ml of NGF).
188
24. Dilute, in other plates 1 ml of cell suspension in 5 ml of medium to continue with the
culture (proliferating cells).
25. Cells induced with NGF will be fixed every 24 hr to study Thy-1 appearance during the
differentiation process. See protocol for IIF for Thy-1.
NOTE: Sodium selenite is highly toxic. Handle carefully.
DIFERENCIATION OF CAD CELLS
26. Grow cells CADcells in DMEM-F12 medium with Pen/Strep, 8% FCS.
27. Obtain confluent cells in 6-well plate to keep a stock of cells growing.
28. Obtain confluent cells in a 10cm plate for the experiment
29. Recover cells from the 10cm plate and resuspend them in 5ml of medium. Pass them
through a sterile needle (syringe) to separate cells and count them.
30. Plate 200.000 cells per well in a 6-well plate in medium in the absence or presence of
8% FBS or 50 ng/ml of sodium selenite (selenite stock is prepared at 10g/ml in sterile
medium without FBS, aliquoted in 30 l aliquots and stored frozen at 20C).
Wells 1 to 3 are of proliferating cells (medium with 8% FBS)
31.
Wells 4 to 6 do not have FBS and contain 50ng.ml of
sodium selenite to induce differentiation
1
3
2
32.
Recover cells from 2 wells (wells 1 and 4) after 48 hr
incubation. Rinse them once with sterile PBS
4
5
6
33.
Place them in an eppendorf tube and spin them in
microcentrifuge at 2000 rpm for 5min. Label the tube well to identify at the end
differentiated vs non-differentiated cells and the number of days they have been in
culture.
34. Remove all liquid without touching the pellet of cells. Loose up the pellet.
35. Add Laemli buffer (1x) containing 5% beta-mercaptoetanol. Use approx. 80l of
sample buffer per half a million cells
36. Vortex the cells well. Boil them for 3 min
37. Store solubilized cells at 20C until all samples are ready to be run on a gel
38. At day 3 change medium to the cells fofr the same they already have
39. Day 4, Recover cells from 2 wells (wells 2 and 5). Rinse them once with sterile PBS.
Repeat steps 8 to 12.
40. Day 6 recover cells from the next 2 wells (wells 3 and 6). Rinse them once with sterile
PBS. Repeat steps 8 to 12.
41. Run the samples in a 12% gel and do immunoblot as indicated (protocol for gels and
blots)
189
DIFERENCIATION OF PC12 CELLS

43. Obtain confluent cells in 6-well plate to keep a stock of cells growing.
44. Obtain confluent cells in a 10cm plate for the experiment
45. Recover cells from the 10cm plate and resuspend them in 5ml of medium. Pass them
through a sterile needle (syringe) to separate cells and count them.
46. Plate 200.000 cells per well in a 6-well plate in medium with serum in the absence or
presence of 10 or 50 ng/ml of NGF. (NGF stock is prepared at 10g/ml in sterile
medium containing 10% FBS, aliquoted in 30 l aliquots and stored frozen at 20C).
Wells 1 to 3 are of proliferating cells (NO NGF)
47.
Wells 4 to 6 have NGF to induce differentiation
48.
Recover cells from 2 wells (wells 1 and 4) after 48 hr
1
3
2
incubation. Rinse them once with sterile PBS
49.
Place them in an eppendorf tube and spin them in
4
5
6
microcentrifuge at 2000 rpm for 5min. Label the tube
well to identify at the end differentiated vs non-differentiated cells and the number of
days they have been in culture.
50. Remove all liquid without touching the pellet of cells. Loose up the pellet.
51. Add Laemli buffer (1x) containing 5% beta-mercaptoetanol. Use approx. 80l of
sample buffer per half a million cells
52. Vortex the cells well. Boil them for 3 min
53. Store solubilized cells at 20C until all samples are ready to be run on a gel
54. At day 3 change medium to the cells fofr the same they already have
55. Day 4, Recover cells from 2 wells (wells 2 and 5). Rinse them once with sterile PBS.
Repeat steps 8 to 12.
56. Day 6 recover cells from the next 2 wells (wells 3 and 6). Rinse them once with sterile
PBS. Repeat steps 8 to 12.
57. Run the samples in a 12% gel and do immunoblot as indicated (protocol for gels and
blots)
190
TREATMENT OF EL-4-F CELLS WITH G418

16. Count cells.
17. Take 25 x 106 of EL-4-f cells . Spin the cells (2000 rpm, 5 min, RT) and resuspend
them in warm 10% FCS/RPMI/5mM -mercaptoethanol/antibiotics (CM), at 107
cells/ml.
18. Prepare 3 petri dishes as follows:
dish#
Add
1__________________________________________
2__________________________________________
3__________________________________________
19. To each plate add cells for a final concentration of 0.25 x 106 cells/ml. Leave them in
incubator (37C/CO2) overnight.
NEXT DAY....................
20. Count cells...
dish#
Number of cells*
%dead cells
1__________________________________________
2__________________________________________
3__________________________________________
*number of cells only consider alive cells.
21. Spin at 2000 rpm 5 min. Discard supernatant.
22. Resuspend the cells in 300 l of CM.
23. Gently overlay the cells over a 800 l of FCS (in eppendorf tubes) Spin 40 seconds at
2000 rpm. Discard supernatant
24. Repeat step 6 and 7 twice
25. Dilute cells to a concentration of 0.25 x 106 cells/ml as follows:
dish#
Add
1__________________________________________
2__________________________________________
3__________________________________________
26. Incubate at 37C/ CO2 overnight.
NEXT DAY..................(repeat same as last day)
27. Count cells...
dish#
Number of cells*
%dead cells
1__________________________________________
2__________________________________________
3__________________________________________
*number of cells only consider alive cells.
28. Spin at 2000 rpm 5 min. Discard supernatant.
29. Resuspend the cells in 300 l of CM.
30. Gently overlay the cells over a 800 l of FCS (in eppendorf tubes) Spin 40 seconds at
2000 rpm. Discard supernatant
31. Repeat step 6 and 7 twice
32. Dilute cells to a concentration of 0.25 x 106 cells/ml as follows:
dish#
Add
1__________________________________________
2__________________________________________
3__________________________________________
191
33. Incubate at 37C/ CO2 overnight.

Test cells by FACS analysis to check expression of Thy-1 and CD3 and in a stimulation
assay to check PY phosphorylation.
192
METHOTREXATE PREPARATION
-- Be very careful when preparing this solution because it is highly toxic.

-- Solubilize 25 mg in 10.2 ml of milliQ water.
-- Neutralize solution with 1N NaOH (final pH ~7.0-7.4).
-- Sterile filter and aliquot it in sterile eppendorf tubes (500 l/tube).
-- Store at -20C.
193
THAWING THE CELLS

-- Thaw 1 vial quickly by agitating it at 37C. Transfer cells to a sterile 15 ml Falcon tube.
-- Spin the cells down at 2500 rpm, room temperature, 3 min.
-- Withdraw and discard medium and add 1 ml of fresh medium. Gently agitate tube to
resuspend cells and plate them out in a 10 cm petri dish with 10 ml of medium.
-- Add 100 l of Methotrexate solution, mix carefully and leave cells in the incubator.
These cells are fibroblasts. They grow slowly and they can take as much as a week to reach
confluence. Leave them in the incubator for a couple of days and then watch them whether
they look good or not. If they are not too happy you may try replacing the medium
(medium+ methotrexate), by spinning them down if they are not stack or by withdrawing
and adding if they look stack to the plate ( they look flat when stack and round when loose)
194
FREEZING CELLS
To freeze cells grow them to about 50-60% confluency starting from 1:10 dilution. Then
treat cells as follows:
-- take medium away, wash cells with 2 ml of trypsin solution (0.25% Hanks buffered
saline).
-- remove immediately and replace with 2 ml fresh trypsin; wait until cells come off bottom
of plate, then immediately add 2 ml of medium.
-- pipette up and down a few times, spin down in blue cap Falcon tube.
--remove supernatant and replace with 4 ml of freezing solution (10% DMSO + 90%
medium).
-- Vortex lightly until cells go into solution.
-- Make 4x 1 ml aliquots in screw cap-tubes; freeze at -70oC in styrepor box over night,
then store away in liquid nitrogen.
195
ADAPTATION OF CELLS TO SERUM FREE MEDIUM (SFM)

SEQUENTIAL ADAPTATION
1.Subculture the cells growing in serum-supplemented medium into a 1:1 (v/v)
mixture of SFM and serum supplemented medium.
2.When the cell density is >5 x 105 cells/ml, subculture the cells in an equal volume
of SFM at a cell density 2.5 x 105 to 3 x 105 cells/ml.
3.Continue to subculture after the cell density is >5 x 105 cells/ml in SFM until the
serum is 0.1% with about 85% cell viability.
4.Subculture the cells into SFM with an inoculum of 2.5 x 105 to 3 x 105 cells/ml.
5.When the cell density is 1 x 106 to 3 x 106 cells/ml (4 to 6 days post planting)
subculture the cells again.
6.Stock cultures of SFM adapted cells should be subcultured in SFM every 3 to 5
days when the cell density is 1 x 106 to 3 x 106 cells/ml with 90% viability.
DIRECT ADAPTATION
Some cells can be directly adapted from serum containing medium to SFM. For direct
adaptation, the cell inoculum should be 1.5 x 105 to 3 x 105 cells/ml. Cells should be
subcultured when the cell density is 1 x 106 to 3 x 106 cells/ml. Cells are fully adapted
to SFM when the cell density is 2 x 106 to 4 x 106 cells/ml after 4 to 7 days in culture.
Stock cultures of cells adapted to SFM should be subcultured in SFM every 3 to 5 days
when the cell density is 1 x 106 to 3 x 106 cells/ml with 90% viability.
196
HOECHST STAINING
Method
Grow cells on cover slips until 80% confluent, then wash 3x with PBS.
Dehydrate samples in methanol for 10min at RT and subsequently rehydrate slowly as
indicated:
25) place cover slips on wet paper towels in a humid chamber at 4oC to store cells for
longer periods of time.
26) Place cover slips in a bath of 70% ethanol for 1min, followed by a bath of dest. water
for another minute.
Stain with an aqueous solution of 1g/ml Hoechst 33258 (bisbenzamidine) for 30min at RT
in darkness. Use 1ml of reagent per well in a 6-well plate or 0.5ml per well in a 24-well
plate.
Remove Hoechst stain by aspiration and wash 2x with nanopure water.
Mount cover slips in glycerol:H2O (1:1) or in DAKO fixation medium.
Look at samples under UV using 346nm excitation and 460nm emission wavelength.
Nuclei are visible as pale blue fluorescent stuctures within cells.
Established in Chile by Claudio Hetz, July 2001
197
PREPARATION OF MONONUCLEAR CELLS FROM HUMAN PERIPHERAL

BLOOD
Blood samples treated with an anti-coagulant are gently overlaid on a saccharose solution
containing sodium diatrizoate and polysucrose (Histopaque 1077, density 1,077 g/ml;
Sigma Chemical Co. N Cat. 1077-1) at a final ratio of 2:1. Samples are then centrifuged in
a clinical centrifuge at 410xg for 30 min (1800rpm) without using breaks at the end of the
run. The opaque interphase containing mononuclear cells is slowly withdrawn (using a
glass pipette) and washed once in PBS followed by centrifugation 10 min at 1500 rpm
(280xg) and twice in PBS followed by centrifugation 10 min at 1000 rpm (125xg). In cases
where separation from red blood cells was not optimal, those are lysed by differential buffer
(Red Blood Cell Lysing Buffer. Sigma Cell Culture; Sigma Chemical Co. N Cat. R-7757)
and mononuclear cells are recovered by centrifugation in sterile PBS. Cells are then
transferred to RPMI 1640 medium supplemented with NaHCO3 2 g/l, 10% heat-inactivated
fetal bovine serum, 50 g/ml L-glutamine and gentamycin (complete RPMI medium). Cell
viability is assessed by the Typan Blue Exclusion method.
Isolation of Monocytes
10-20x106 mononuclear cells are cultured in a 10 cm dish with complete RPMI medium.
After 2h in the incubator, non-adherent cells (lymphocytes) are gently removed. Adherent
cells (monocytes) are washed very gently with sterile PBS to remove all lyphocytes and
then culture is continued in RPMI complete medium.
Differentiation of macrophages and induction of COX2
Monocytes in RPMI complete medium are treated for 3d with 10nM TPA. Cells are then
washed very gently with sterile PBS and incubation is continued for at least another 24h in
RPMI complete medium. COX2 expression is induced in these cells by treatment for 24h
with 50 ng/ml LPS (Lipopolysaccharide, Sigma Cell Culture; Sigma Chemical Co. N
Cat.L-7895)( LPS from any bacteria serotype may be used).
Reference for differentiation of macrophages and induction of COX2:
Plant, M.H. and Laneuville, O. 1999. Characterization of novel transcript of prostaglandin endoperoxide H
synthase 1 with a tissue-specific profile of expression. Biochem. J. 344, 677-685.
Reference for preparation of mononuclear cells:
Montoya, M. Efectos in vitro de bacterias fijadas sobre la actividad citoltica NK humana. Tesis para la
obtencin del grado de Licenciado en Bioqumica. Universidad de Santiago de Chile. 1998.
198
CELL VIABILITY ASSAY (MTS)
Method
Place 1x 104 to 1 x105 cells in 100 l volume in a 96-well plate (Number of cells and timing
of assay need to be determined individually for the respective cells). Add 20 l of
MTS/PMS (100 l of PMS and 2ml of MTS). Incubate for 1-4h at 37oC in cell incubator
until yellow colour is appropriate for measurement. Read values in ELISA reader
(Immunology or Parasitology) at 492nm. Also measure a background value for the reagent
and buffer without cells. This should be deducted from other values. Measurements are to
be made in triplicate for each sample.
Note: Is better to work in sterile conditions
Based on protocol developed in Switzerland by Martin Hunn
Established in Chile by Patricio Rojas
199
USE OF A HEMACYTOMETER
P.J. Hansen
Dept of Animal Sciences, University of Florida
A hemacytometer (also spelled hemocytometer) is an etched glass chamber with raised
sides that will hold a quartz coverslip exactly 0.1 mm above the chamber floor. The
counting chamber is etched in a total surface area of 9 mm2 (see Figure 1).
Figure 1. Dimensions of a hemacytometer.

Calculation of concentration is based on the volume underneath the cover slip. One large
square (see W in Figure 2) has a volume of 0.0001 ml (length x width x height; i.e., 0.01
cm x 0.01 cm x 0.001 cm).
200
MTT METHOD FOR THE EVALUATION OF MIGRATORY CELLS

1.- Once the migration has finished add 10 l 5 mg/ml MTT in the superior compartment
and 80 l in the inferior. Incubate for 2 hours.
2.- Once the incubation has finished, supernatant media are transferred to labeled
eppendorf tubes.
3.- Incubate both compartments with 0.05%, 1mM Tripsin/EDTA in PBS for 15 minutes
at 37C. Add 200 l to the superior compartment and 800 l to the inferior one.
4.- Collect the released cells in the superior chamber with 2 washes with PBS and transfer
them to the correpondant tube of step 2.
5.- The cells that are in the filter or that have passed through it are released with the same
tripsin/EDTA added in the inferior chamber. After collection, they are transferred to the
correspondant tube. They are centifuged in a microfuge. Discard the supernatant and wash
the pellet twice with PBS.
6.- Finally, let the tubes dry. The cristal formed are dissolved with a 3:2 DMSO/isopropanol
mixture shaking and incubating at 37C for 10 minutes.
7.- Read in a ELISA reader at 510 nm.
201
MCF-7 CELLS MIGRATION ASSAY

(Nguyen D.H.D. et al J. Cell Biol. 146:149-164, 1999)
1.- For short time migratory assays or for those using low migration cells it i recommended
to cover with serum only the inferior face of the membrane. To do so, Transwell inserts
membranes (6.5 mm, 8.0 m pore, Costar) are incubated wi 20% FS medium for 2 hours at
37C. After, they are blocked with 10 mg/ml BSA.
2.- Transfer 105 cells in 100 l of medium to the superior compertment. For u-PA treated
cells, these should be pretreated in suspension for 30 minutes. Inhibitors or antibodies
should be added to the cells 15 minutes before the treatment with u-PA. The migration
assay is performed in presence of inducers, inhibitors or antibodies. The inferior
compartment has to have always 800 l of 10% Sf medium.
3.- For the assay in which migration is estimulated with 10 nM u-PA the reagent must be
present in both compartments of the insert.
4.- Once the assay is finished (minimum of 6 hours), the cells must be removed from the
superior compartment with a cotton ear-stick (cotonito).
5.- Cell migration to the inferior compartment is evaluated fixing the membrane in
methanol and staining the cells with 0.1% violet cristal. After staining the cells, the dye is
dissolved with 10% acetic acid to measure the absorbance at 600 nm of the eluate.
6.- For really migratory cells, both sides of the membrane are covered with 20% FS
medium. No FS is added in either compartment of the insert for this case.
202
GROTH IN SOFT AGAR

Test purpose:
To check the ability of the cells (HT-29, C14, C5) to grow in a semisolid medium. It is the
in vitro assay of choice to evaluate cell transformation. To analize the results, you should
consider how many cells out of 100 cells, form colonies. In some cases it is also important
to consider the number of cells that comprise the colonie.
Materials and solutions:
Agar, DIFCO laboratories (0142-15-2), Culture medium (dust), bicarbonate, 100X PSN,
FBS, IPTG or inductors or inhibitors required to perfor3m the experiment, sterile PBS. 1%
Violet cristal stock solution in ethanol, 15 ml Falcon tubes and sterile 1.5 ml eppendorf
tubes.
Biological material: Cells to study, transformed cells as positive control and nontransformed cells as negative control. For example: tumor cells HT-29/cav-1 clone 14, HT29/placIOP clone 5 (transfected with the plasmid containing caveolin-1 and with the empty
plasmid respectively), A 10-IPTG and A10+IPTG as negative and positive controls
respectively1 2.
Equipment: Termoregulated water bath and incubator.
Media and solutions preparation:
2X-20% FBS Medium: To prepare this medium you have to add 1 package of powder to
prepare 1 litre of 1X medium, 3.7 g of bicarbonate and 10 ml of 100X PSN in 500 ml of
nanopure water (half of the indicated volumen for preparing 1X medium). Follow the usual
procedure. Supplement the medium with serum at a final concentration of 20%.
1% Agar solution: weight the appropriate quantity of agar and dissolve in nanopure water.
Autoclave just following preparation. After autoclaving keep the solution at 46C (the agar
remains liquid at this temperature). You can store the solution in the fridge (4C) if you are
not going to use it the same day.
Staining: From the 1% violet cristal stock solution in ethanol, prepare a 0.005% violet
cristal solution in PBS. Keep it at 4C.
Six well plates protocol.
Six well plates protocol.
It is worth mentioning that every cell line has been transformed for their culture. Thus, a nontransformed cell
line has to be chosen as a negative control.
2
Cell produce growth factors, being necessary to grow them at low dilutions.
203
The area of the well is 9.62 cm2. Values must be modified on a well size basis (cylinder
volumen= circunference area x height).
First coat preparation (this coat is 0.5% agar):
1 part 2X medium (1.1 ml) : 1part agar solution (1.1 ml). Final volumen: 2.2 ml
Briefly:
1.- Dissolve agar in a water bath at 80-100C. Once dissolved, cool until 46C.
2.-Transfer the necessary medium volumen for the number of assays to be performed, to 15
ml tubes.
3.- QUICKLY add an equal volumen of 1% agar solution at 46C. Mix thoroughly up and
down without making bubbles. Add 2.2 ml to each well.
4.- Add the necessary inductor volumen so it reaches the desired concentration in 2.2 ml
NOTE: this coat can be stored at 4C for a week. It is not reccomended to store it for longer
periods.
Second coat preparation (this coat is 33% agar):
0.33 parts 1% agar solution (450 l) : 0.33 parts 2X medium (450 l) : 0.33 parts 1X
medium (450 l) Final volumen of the coat= 1.350 ml
Cells must be seeded in normal culture medium one day or more before the assay is
performed. It is necessary to preinduce them. It is recommended that the day of the assay,
the cells should be at a low confluence (20% to 40%) so it will not be necessary to dilute
them so much (a few cells are necessary for the assay)
1.- Melt the agar and keep it at 46C
2.- Wash the cells 2X with PBS. Tripsinize, collect and count the cells.
3.- Centrifuge to pellet the appropriate number of cells (i.e. 5000 for HT-29)4 in 1.5 ml
eppendorf tubes, at 2000 rpm for 5 minutes.
NOTE: As the pellet is very small, take the supernatant out of the tube with a Pasteur
pipette IMMEDIATELY and thoroughly.
4.- Resuspend the adecuate number of cells in 450 l of 2X 20% FBS medium. Pipette up
and down and vortex. Add 450 l of 1X 10% FBS medium and IPTG or the appropriate
treatment at an adecuate concentration to a final medium volumen of 1350 l.
Reccomendation: use cold media so as to compensate the agar temperature.
The number of cells to seed depends on the cell type. thgis parameter has to be determined by the researcher
204
5.- Add to each eppendorf -one by one- 450 l of agar solution. Mix twice up and down
with a micropipette and add onto the first coat CAREFULLY by the side. Do it QUICKLY
to avoid solidification of the agar5.
6.- Incubate in an incubator at 37C, 5% CO2, 80% humidity for 10 days6. It is necessary to
add just a few drops of 2X culture medium (+ inducer) each 3-4 daysto prevent the agar
from drying.
7.- The tenth day collect the assay and stain with 500 l of 0.005% cristal violet solution.
Incubate over night at 4C 7. Check the assay the following day in a Motic
stereomicroscope with both incident and transmitted light, at 40X magnification. Take three
or more representative pictures of each well. For the analysis: adding colonies and
individual cells represents the initial quantity of seeded cells per field.
colony % = colony number/(colony number + individual cells number)
Modified 19.07.01 Pamela Lisboa
No. of cell lines used
No. of assays (+/- treatment)
1% Agar - First layer
1% Agar - Second layer
Total Agar
1X DMEM medium H-glu
2X DMEM medium first layer
2X DMEM medium second layer
2X total DMEM medium
1
2
1
1.1
0.45
1.55
0.45
1.1
0.675
1.775
2.2
0.9
3.1
0.9
2.2
1.35
3.55
4
8
8.8
3.6
12.4
3.6
8.8
5.4
14.2
6
12
12
12
24
5.4
18.6
5.4
13.2
8
16
17.6
7.2
24.8
7.2
17.6
10.8
28.4
It is important to distribute a homogeneous coat so you have a homogeneous experiment that gives you
reproducible results when you observe different fields.
6
This parameter has to be determined by the researcher for a particular cell line and assay conditions.
7
Staining allows you to distinguish among individual cells otherwise difficult to observe.
205
G: PROTEIN FUSION
GST ASSAY........................................................................................................................207
GST FUSION PROTEIN EXPRESSION...........................................................................208
GST FUSION PROTEIN PURIFICATION........................................................................209
MATRIX PREPARATION FOR MALTOSE FUSION PROTEIN PURIFICATION
(MANUFACTURER`S PROTOCOL)................................................................................210
MALTOSE FUSION PROTEIN PURIFICATION (MANUFACTURER`S PROTOCOL)
.............................................................................................................................................211
MALTOSE FUSION PROTEIN PURIFICATION.............................................................212
MICROCONCENTRATION OF GST FUSION PROTEINS USING CENTRICONS.....213
GROWTH OF BACTERIA EXPRESSING GST-FUSION PROTEINS...........................214
PURIFICATION AND SOLUBILIZATION OF INCLUSION BODIES..........................215
REGENERATION OF GSH-AGAROSE AFFINITY MATRIX........................................216
206
GST ASSAY
October 1992
Based on Habig, W. H., Pabst, M. J., and Jakoby, W. B. 1974. Glutathione S-Transferases:
The first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249(22):7130-7139.
In a 1 ml quartz cuvette mix:
40 l 25 mM reduced glutathione
40 l 25 mM (5mg/ml)1-chloro-2,4-dinitrobenzene in ethanol
50 l 1 M phosphate buffer, pH 6.5
q.s H2O to 1 ml (including volume of enzyme to be added)
Scan at 340nm for 30 or 60 sec to record background rate. Add enzyme and mix. Scan
again to record the reaction rate.
Calculations:
= 9.6 mM-1cm-1
207
GST FUSION PROTEIN EXPRESSION

STARTING
MATERIAL: PKC construct:
Culture Vol.:
Harvest date:
Expression:
E. coli strain:
Expression temp./time:
PROTOCOL: Start from a single colony on agarose plate with

amp/.........)
Date when colonies were plated:
antibiotic
selection
(+
Innoculate 3 ml LB (+amp/..........) over night with streak from single colony

at 37C.
Time A600 add.
Start next day at 37C. Per 50 ml culture volume, use 1 ml o/n to
innoculate. Conical flask for expression must be at least 5x larger than culture volume to
allow adequate aeration of E. coli during growth and expression. Grow culture to OD 600 of
0.5 (subtract background with LB medium). Transfer culture to 20C (aquatron in cold
room,150 rpm) and continue growth until OD600 of 0.7-0.75 is reached. Induce expression
by
addition of IPTG to a final concentration of 0.1-0.2 mM.
If construct is in protease defficient BL21, expression takes about 6
- 8 hours; if in XL1B expression goes o/n. Final OD should be 1.2-1.4. Depending on the
construct being expressed, particularly when using BL21 cells, these conditions may be
modified.
Put 50 ml of culture medium in Falcon tube; spin 10 min. at 5000
rpm in clinical centrifuge to collect E. coli pellet. Discard supernatant. Invert Falcon tubes
on paper and let drain for 5 min. Freeze pellets at -20C.
208
GST FUSION PROTEIN PURIFICATION

START MAT.:
constuct:
culture vol.:
harvest date:
E. coli strain:
Expr. time/temp.:
harvest Nr.:
EXT.BUFFER: to 50 mM HEPES, pH 8.0; 10% ethylene glycol

add 0.2% TX-100(10% stock);1mM PMSF(100X =100mM)
1x BHT/DTPA (1000x BHT= 5 mg/ml; DTPA = 500mM)
0.5 mg/ml Lysozyme (100x = 50 mg/ml)
1x BAL (1000x is 10 mg/ml benzamidine, 2 mg/ml
antipain, 1 mg/ml leupeptin)
SONICATION:
1 X 20 at power level 1, constant pulse; or 1x
cavitation 5 min.
GST activity in 10 l:
A340/min
Values should be around 0.8 to 1.2. If not repeat
sonication/cavitation step as above.
A340/min
CENTRIFUGATION: at least15 min. in Eppendorf centrifuge at 13000 rpm.
Supernatant should be opalescent with only a week yellow/brown colour. If colour is
strong, repeat spin. Pool supernatants.
Supernatant volume:
ml
A340/min
Is activity good? Is pH 8 ? Purify on GSH column.
PURIFICATION: Equilibrate column in 0.1x PBS (AQs version).
Load supernatant on column 3x.
GST act. in 10l of flow through:
A340/min
How much has bound to column? Is binding good?
Wash column with 30 ml of 0.1x PBS (AQs version).
Elute fusion proteins with fresh Elution buffer containing 10 mM reduced GSH in
50 mM HEPES pH 8.0 with 10% ethyleneglycol. Add elution buffer 1 ml at a time. After
first ml has run through, cap column add second ml and wait 15 min. Then remove cap and
continue with elution.
Fraction Nr.
1
2
3
4
5
6
7
GST act.
(A340/min)
est. protein (mg/ml)
Amido Schwarz protein (mg/ml)
Aliquot size (ml)
209
MATRIX PREPARATION FOR MALTOSE FUSION PROTEIN PURIFICATION

(MANUFACTURER`S PROTOCOL)
Description of matrix:
The matrix (amylose resin: cat # 800-21S, Biolabs) supplied preswollen in 30%
ethanol. Take 2 ml of amylose resin and place it in a column (Cat # 731-1550, BioRad.).
After washing with five column
volumns of buffer (20 mM Tris-HCl pH7.4, 0.2 M,
NaCl, 10 mM -mercaptoethanol, 1 mM EDTA), matrix is ready for use.
Column buffer:
Per litre/ 20 ml 0.5M sodium phosphate , pH7.2, 29.2 g NaCl, 1 ml 1M sodium
azide, 0.7 -mercapto-ethanol (optional), 1 ml 1M EGTA pH7 (optional). Adjust to pH7,
if necessary
Regeneration:
The packed resin may be regenerated with the following washes:
ddH2O: 3x column volume
ddH2O: 1x column volume
column buffer: 3x column volumes
Storage:
The used resin should be stored in column buffer+0.02% sodium azide at 4C.
210
MALTOSE FUSION PROTEIN PURIFICATION (MANUFACTURER`S

PROTOCOL)
START MAT.:
constuct:
culture vol.:
harvest date:
E. coli strain:
Expr. time/temp.:
Harvest Nr.:
EXT.BUFFER:
5 ml of lysis buffer: 10mM Na2HPO4, 30 mM NaCl,

0.25% Tween 20, 10 mM -ME (optional), 10 mM EDTA (pH8), 10
mM EGTA(pH7).
Ajust to pH7.0.
Freeze sample in a dry ice-ethanol bath. Thaw in cold water.
SONICATION:
CENTRIFUGATION:
at least 20 min. in Eppendorf centrifuge at 13000rpm, and 4C. Keep
samples on ice
PURIFICATION: Equilibrate column in washing buffer

Load supernatant on to column 3x at 4C
How much has bound to column? Is binding good? (check
by WB analysis)
Wash column 10 column volumes of washing buffer (20
mM Tris-HCl pH7.4, 0.2 M NaCl, 10 mM -mercaptoethanol, 1 mM EDTA) Elute fusion proteins with fresh Elution
buffer containing 10 mM maltose in washing buffer. Add
elution buffer 1 ml at a time. After first ml has run through,
cap column add second ml and wait 15 min. Then remove
cap and continue with elution 1 ml a time
Fraction Nr
Aliquot size (ml)
211
MALTOSE FUSION PROTEIN PURIFICATION
START MAT.:
constuct:
culture vol.:
harvest date:
E. coli strain:
Expr. time/temp.:
Harvest Nr.:
EXT.BUFFER:
to 50 mM HEPES, pH 8.0; 10% ethylene glycol

add 0.2% TX-100 (10% stock) ; 1mM PMSF (100X=100mM)
1x BHT/DTPA (1000x BHT= 5 mg/ml; DTPA = 500mM)
0.5 mg/ml Lysozyme (100x = 50 mg/ml)
1x BAL (1000x is 10 mg/ml benzamidine, 2 mg/ml
antipain,
1 mg/ml leupeptin)
FRENCH PRESS: 3 times

CENTRIFUGATION:
At least 15 min. in Eppendorf centrifuge at 13000 rpm.
Supernatant should be opalescent with only a week yellow/brown
colour. If colour is strong, repeat spin. Pool supernatants.
PURIFICATION: Equilibrate column in 0.1x PBS (AQs version).
Load supernatant on column 3x
How much has bound to column? Is binding good?
Wash column with 30 ml of 0.1x PBS (AQs version).
Elute fusion proteins with fresh Elution buffer containing 10 mM
maltose in 50 mM HEPES pH 8.0 with 10% ethyleneglycol. Add
elution buffer 1 ml at a time. After first ml has run through, cap
column add second ml and wait 15 min. Then remove cap and
continue with elution.
Fraction Nr

Aliquot size (ml)
212
MICROCONCENTRATION OF GST FUSION PROTEINS USING CENTRICONS
Wash microconcentrator 30 (Amicon; Cat.Nr 42409) with distilled water. Spin 10 min at
4C, 13000 rpm.
Incubate 10 min with 0.1% PVP (Polyvinylpyrrolidone 30 [Fluka; Cat.Nr. 81422] or 40
[Sigma; Cat.Nr.PVP-40]) diluted in distilled water.
Rinse and wash the microconcentrator with distilled water 10 min, 13000 rpm at 4C.
Add 500 l of GST fusion protein and 10 l of PVP 1%. Spin 15 min, 13000 rpm at 4C.
Keep the flow through. Repeat.
To elute, invert the microconcentrator and insert into an eppendorf-like tube provided.
Collect concentrated sample by brief centrifugation in a clinical centrifuge after adding 100
l of Hepes Buffer pH8 with protease inhibitors (BAL/see preparation of BAL 1000x stock;
and PMSF/ 1mM)
213
GROWTH OF BACTERIA EXPRESSING GST-FUSION PROTEINS

October 1993
After a construct has been isolated a glycerol stock should be made. From this stock new
plates may be streaked whenever it is necessary to grow a batch of protein. If you
are growing pGEX in XL1Blue, it may be grown on LB+Amp+Tet, if in BL21 or
DH5, then LB+Amp only. From the plate select a single colony and grow a 10 ml
culture overnight at 37 C, with shaking.
In the morning innoculate a flask of the appropriate size to an OD 600 of 0.15-0.25 (This is
total OD600, LB media as we make it provides a background OD 600 of approximately
0.07-0.10). This is about 2 ml innoculum for 125 ml fresh media. Place in 37 C
shaker until OD600 is 0.5. Different cell lines grow at different rates so follow your
cells carefully until you have a feel for the rate of growth. If the cells have grown
too dense, it is possible to dilute them to the correct density, as long as they are still
in log phase growth. At OD600 0.5, transfer the flasks to a 20 C shaker bath and
allow to grow to an OD600 of 0.75. In our lab, we use a shaker with water but no
heat to maintain a temperature of approximately 20 C. At OD 600 0.75 induce with
1-2 M IPTG, and if it is a Zn-containing protein, we also add 1 M ZnCl2 .
Harvest the bacteria when a density of OD600 1.2-1.4 is reached. For XL1Blue this requires
an incubation of approximately 16 hours, for BL21 this density may be reached
after only 8 hours of culture.
Notes about growth conditions for various constructs: Constructs that are predicted to
be short and highly water soluble may be grown at 37 C in just a few hours. The
rationale for growing at lower temperature is to slow growth and allow correct
protein folding to occur. Under fast growth conditions inclusion body formation is
favored. For some protein constructs, inclusion body formation may be valuable
and allow easy purification. But for those cases in which protein reconstitution is
not practical or productive, then adjusting growth conditions may permit the
expression of the protein as a soluble molecule. We tried growing at 37, 30 and 20
C, as well as in the presence and absence of 10% glycerol. We have had almost
universal success generating soluble, full-length proteins in either BL21 or
XL1Blue cells growing at 20 C without glycerol.
214
PURIFICATION AND SOLUBILIZATION OF INCLUSION BODIES

February 1991. Biotechniques (1991) 6:748-753.
For a cell pellet from 1 liter of cells:
Remove outer membrane:
1) Suspend cells in 50 ml Buffer A:
20 mM Tris-Cl, pH 7.5
20 % sucrose
1 mM EDTA
2) Incubate on ice 10 min
3) Centrifuge 4000 xg 10 min. Discard sup.
4) Resuspend pellet in 50 ml ice cold water for 10 min.
5) Centrifuge 8000 xg 10 min. Discard sup.
6) Resuspend pellet (spheroplasts) in 10 ml Buffer P:
PBS (-Ca++, -Mg++)
5 mM EDTA
1 g/ml leupeptin
20 g/ml aprotinin
0.5 mM PMSF
Lyse cells:
7) Sonicate cell suspension 50 W 3 x 30 sec on, 30 sec off
8) Add
1.3 x102 U/ ml RNase T1
40 g/ ml DNase I
9) Incubate room temperature 10 min
10) Dilute cell suspension with 40 ml Buffer P
11) Centrifuge 13,000 xg 20 min. Discard sup.
Wash inclusion bodies:
12) Resuspend pellet (inclusion bodies) in 4o ml Buffer W:
PBS (-Ca++, -Mg++)
25% sucrose
5 mM EDTA
1% Triton X-100
13) Incubate on ice 10 min
14) Centrifuge 25,000 xg 10 min. Discard sup
15) Repeat for a total of 3 washes
Denaturation and renaturation of the inclusion body protein:
16) Resuspend the washed pellet in 10 ml Buffer D
50 mM Tris-Cl, pH 8
5 M guanindinum chloride
5 mM EDTA
17) Sonicate 5 sec 50 W (this must be gentle)
18) Incubate the sonicated suspension on ice 60 min
19) Centrifuge 12,000 xg 30 min
20) Mix the sup with 100 ml Buffer R:
50 mM Tris-Cl, pH 8
1 mM DTT
20% glycerol
21) Stir gently overnight at 4 C
22) Centrifuge 13,500 xg 30 min. The supernatant is the pure refolded protein.
215
REGENERATION OF GSH-AGAROSE AFFINITY MATRIX

After multiple usage (5-10 preparations) the affinity columns with 2-3 ml matrix volume do
not work as well any more and need to be cleaned up.
This is done in the following way:
First wash with 20-30 ml of 0.1M Borate pH8, containing 1M NaCl.
Rinse with 20-30 ml dest. water
Next, wash with 20-30 ml of 0.1M Acetate pH 4.0, containing 1M NaCl.
Rinse again with 20-30 ml dest. water.
If the column has accumulated a lot of DNA, which is often the case when working
with constructs expressed in BL21/DE3 cells, an additional step may be added here.
Rinse column with 20-30 ml of 20 mM Acetate pH 5.0, 10 mM MgCl 2. Then add 23 ml of DNase at final conc. of 50 ug/ml. Cap column and incubate at RT for 1 hour.
Rinse again with 20-30 ml of dest. water.
Now the column can be rinsed with 20-30 ml of 0.1x PBS containing 5mM DTT or b-ME
and is ready to be used for affinity purifications. This step should always be done if the
column has been stored for a while.
Alternatively, the column can be stored in 1M NaCl with 0.02% thimerasol. Make sure
column does not dry out - check from time to time. Another possibility is to relyophilize the
gel matrix in the presence of lactate (check with Sigma).
DNase Stock: Dissolve DNase (Boehringer Mannheim #104132; 20000U or 2.78mg) at
final conc. of 1mg/ml by adding 1.3 ml dest water and 1.6 ml conc. glycerol. Mix and make
100 ul aliquots that are stored frozen at -20C. Each aliquot is sufficient for the regeneration
of a column.
AQ 6-1-95.
216
H: MOLECULAR BIOLOGY
ALKALINE PLASMID PREP...........................................................................................218
BACTERIAL MEDIA (MANIATIS)..................................................................................218
CDNA SYNTHESIS FROM MRNA AND PCR AMPLIFICATION................................218
MAKING COMPETENT CELLS......................................................................................218
ALKALINE MAXI DNA PREP/CSCL2-GRADIENT.......................................................218
TRANSIENT TRANSFECTION IN COS..........................................................................218
PREPARING AND RUNNING SEQUENCING GELS....................................................218
SAMPLE PREPARATION FOR DSDNA SEQUENCING...............................................218
GEL ISOLATION OF RESTRICTION DIGESTED PCR-DNA.......................................218
LIGATION PROTOCOL FOR DOUBLE DIGESTED PCR-DNA & PGEX2T...............218
PLASMID MINI BOILING PREP.....................................................................................218
MINIPREPARATION OF PLASMID DNA BY ALKALINE LYSIS................................218
NORTHERN ANALYSIS...................................................................................................218
PCR WITHOUT HOT START........................................................................................218
PCR TO CHECK PLASMID DNA FOR INSERTS...........................................................218
ISOLATION OF PCR FRAGMENTS FOR LIGATION...................................................218
ALKALINE PHOSPHATASE TREATMENT OF DNA....................................................218
PROTOCOL TO GENERATE POINT MUTATIONS AND AT THE SAME TIME
RESTRICTION SITES.......................................................................................................218
PROTEINASE K TREATMENT OF PCR FRAGMENTS................................................218
RESTRICTION DIGEST OF PROTEINASE K TREATED DNA....................................218
RESTRICTION DIGEST OF PLASMID DNA.................................................................218
PREPARATION OF MRNA FROM CULTURE CELLS..................................................218
TRANSFORMATION PROTOCOL FOR LIGATED DNA..............................................218
DNA SEPARATION IN AGAROSE GEL.........................................................................218
EXTRACTION AND DNA PURIFICATION FROM AN AGAROSE GEL.....................218
GLYCEROL STOCKS........................................................................................................218
LIGATION OF TWO DNA FRAGMENTS.......................................................................218
MINIPREP OF PLASMID DNA........................................................................................218
PCR AMPLIFICATION.....................................................................................................218
RESTRICTION DIGEST OF PLASMID DNA.................................................................218
STRATEGY TO MAKE A POINT MUTATION WITH A RESTRICTION SITE
INSERTED..........................................................................................................................218
TRANSFORMATION OF BACTERIA WITH A RECOMBINANT PLASMID..............218
217
PCR METHOD TO CHECK BACTERIAL COLONIES OBTAINED FOLLOWING

TRANSFORMATION........................................................................................................218
RNA ISOLATION USING TRI REAGENT.....................................................................218
HUMAN COX-2 SEMI-QUANTITATIVE RELATIVE RT-PCR (AMBION)................218
TRANSFORMATION OF CA-COMPETENT BACTERIA WITH LIGATED DNA.......218
MAKING COMPETENT CELLS USING CACL2...........................................................218
TRANSFECTION BY ELECTROPORATION OF NIH3T3 CELLS................................218
ELECTROTRANSFECTION OF A20 CELLS..................................................................218
ENDOFREE DNA MIDI PREP (R. BILLETAS VERSION)............................................218
ENDOFREE DNA MAXI PREP WITH QIAGEN COLUMNS........................................218
PLASMID DNA MINIPREPS............................................................................................218
DNA MINI PREPS WITH PROMEGA COLUMNS.........................................................218
DNA MIDI PREP WITH QIAGEN COLUMNS...............................................................218
DNA MIDI PREP WITH PROMEGA COLUMNS...........................................................218
DNA MINI PREP WITH QIAGEN COLUMNS...............................................................218
218
ALKALINE PLASMID PREP

This protocol yields large quantities of DNA (up to 200 ug) suitable for sequencing
and basically any other purpose:
1.- Grow 200 ml bacteria in L-broth with antibiotica selection (i.e. 50-200 ug/ml
ampicillin) to stationary phase (i.e. over night).
2.- Transfer to 250 ml bottle; centrifuge in rotor A6.14 at 6000 rpm (6000xg). Water
may be added to balance bottles.
3.- Decant supernatant into original container for decontamination, with chlorox or
red detergent available at BIL. Let centrifuge bottle drain upside down for a few minutes on
paper. Be careful that the pellet does not slip out of the bottle.
4.- Add 5 ml of solution I (GTE). Resuspend by gentle aspiration with a 10 ml
pipette and then transfer to 50 ml polypropylene tube. Vortex until thoroughly suspended.
Place on ice.
5.- Add 1 ml of fresh lysozyme solution at 10 mg/ml GTE.
6.- Incubate 10 min on ice.
7.- Add 1 ml 10% SDS and 1 ml 2M NaOH to 10 ml water and then dispense into
tube with bacteria lysate. Incubate on ice 10 min.
8.- Add 9 ml Solution II (3M NaAc). Mix well but carefully. Let sit on ice for a few
minutes.
9.- Spin at 4C, 13000 rpm (20000xg) in A8.24 rotor for 10 min.
10.- Pour supernatant with plasmid DNA into fresh 250 ml bottle. Add 9 ml
Solution III (8M Ammonium acetate) followed by 70 ml 95% ethanol. Mix well but
carefully. Place at -20C for 30 min. or over night.
11.- Spin at 4C, 8000rpm (.............xg) in A6.14 rotor for 20 min. Decant
supernatant, let drain upside down on paper for a few minutes. Be careful that pellet does
not slip out!
12.- Resuspend pellet in 3 ml Solution IV (Tris/EDTA). Transfer to 15 ml Falcon
tube (polypropylene) and measure volume transferred. Note volume and wash original tube
with an amount that will add up to exactly 4 ml final volume.
13.- Add 4.5 g of CsCl, vortex to dissolve, warm at 37C if neccessary.
14.- Add 0.45 ml EtBr (10 mg/ml in water). Be careful!!!
15.- Incubate on ice for 5 min. Spin 10 min. at 4C, 8000 rpm (7700xg) in A8.24
rotor.
16.- Pipette supernatant into 6 ml Quick-seal type polycarbonate tubes (Kontron
Instruments #9091-90386) under Raymonds bench.
17.- Balance two tubes against one another using CsCl solution (1g CsCl plus 1 ml
219
water) if neccessary. Both tubes should be filled up to the neck - otherwise they collapse!
Tubes must be balanced together with their respective Ultracrimp TM (Sorvall#03999) tube
plugs. Seal tubes with device in common facility hallway below gel dryer.
18.- Insert tubes into TV865 rotor located under dryer. Before attempting to tighten
screw caps, make sure rotor is secured in designated place below gel dryer. To tighten have
indicator on device pointing to right. To loosen caps the next day, indicator should be
pointing to the left. Tighten until device starts clicking.
19.- Take rotor and place on shaft in Kontron Ultracentrifuge (Centrikon T-2070).
Close door and pgm. according to needs. For DNA preps. 15 hr (= , for programming
purposes), 50000 rpm, 15C.
20.- Next day stop machine, remove rotor, loosen caps very slowly and remove
tubes from rotor carefully. Pull DNA band under UV light. Minimize exposure to UV. Poke
small gauge needle into top of Ultracrimp tube to allow pressure equilibration. Pull
supercoiled DNA band roughly in middle with 10 ml syringe and large gauge needle, so as
not to shear DNA.
21.- Band should be quantitatively recovered in about 1 ml volume. Transfer this
volume to 15 ml polypropylene. Add 3 ml of dd water. Extract several times with water
saturated isoamyl alcohol. Centrifuge in clincal centrifuge to separate phases if neccessary.
22.- After last spin withdraw all of the isoamyl alcohol and add 8 ml of 95%
ethanol. Let sit at -20C for 30 min. or overnight.
23.- Centrifuge for 10 min. at 4C, 10000 rpm (12000xg). Wash pellet with 70%
ethanol to remove salt. Drain well. Leave to air-dry on bench or put in vacuum dessicator or
in 37C ventilated incubator.
24.- Dissolve in 0.5 - 1.0 ml TE and measure DNA conc. In 500 ul, conc. will be
around 500 ug/ml. Determine conc. by measuring OD(260nm) according to: OD x 50 (for
ds DNA) x dil. factor (about 200x) = conc. ug/ml. Also check OD260:OD280 ratio which
should be 1.6
plasmid
OD260
OD280
ratio
conc.
Alkaline plasmid prep solutions

Solution I: 50 mM glucose (dextrose), 10 mM EDTA, 25 mM Tris pH8. (4.5 g glucose, 20
ml of 250mM EDTA pH8, 12.5 ml 1M Tris, pH8; fill up to 500 ml with water).
Solution II: 3M NaAc (400 g NaAc in 1l, adjust pH to 4.8 with HAc before bringing up to
final volume).
220
Solution III: 8M Ammonium acetate. Dissolve 62g per 100ml solution.

Solution IV: 50 mM Tris, 2 mM EDTA pH8 (25 ml 1M Tris pH8, 4 ml 0.25 M EDTA pH8
in 500 ml total volume).
Comments
AQ, 2 Nov. 1994
221
BACTERIAL MEDIA (MANIATIS)

from: J. Sambrook, E. F. Fritsch, T. Maniatis: Molecular Cloning; A Laboratory Manual;
2nd edition, Cold Spring Harbor Laboratory Press 1989.
LB Medium (Luria-Bertani Medium)
To 950 ml of deionized H2O, add:
bacto-tryptone
10 g
bacto-yeast extract
5g
NaCl
10 g
Adjust to final volume of 1 liter by adding ddH2O.
NZCYM Medium
NZ amine
10 g
NaCl
5g
bacto yeast extract
5g
casamino acids
1g
MgSO4.7 H2O
2g
NZYM Medium
NZYM mdium is identical to NZCYM medium except that casamino acids are
omitted.
NZM Medium
NZM medium is identical to NZYM medium, ecxept that bacto-yeast extract is
omitted.
SOB Medium
bacto-tryptone
20 g
bacto-yeast
5g
NaCl
0.5 g
Shake until the solutes have dissolved. Add 10 ml of a 250 mM solution of KCl.
(This solution is made by dissolving 1.86 g of KCl in 100 ml of deionized H 2O.)
Adjust the pH to 7.0 with 5 N NaOH (ca 0.2 ml). Adjust the volume of the solution
to 1 liter with deionized H2O. Sterilize by autoclaving for 20 minutes at 15 lb/sq. in.
on liquid cycle.
Just before use, add 5 ml of a sterile solution of 2 M MgCl 2 (This solution is made
by dissolving 19 g of MgCl2 in 90 ml of deionized water. Ajdust the volume of the
solution to 100 ml with deionized water and sterilize by autoclaving).
SOC Medium
SOC medium is identical to SOB medium, except that it contains 20 mM glucose. After the
SOB medium has been autoclaved, allow it to cool to 60 C or less and then add 20 ml of a
sterile 1 M solution of clucose. (This solution is made by dissolving 18 g of glucose in 90
ml of deionized water. After the sugar has dissolved, adjust the volume of the solution to
100 ml with deionized water and sterilize by filtration through a 0.22 m filter.)
222
Source: A. Quest, M. Hunn; personal communication.

LB glycerol (2x)
Dissolve 1 g bactrotrypton (Difco), 0.5 g bacto yeast (Difco) and 1 g NaCl in 66 ml
of sterile H2O. Then add 34 ml of 87% glycerol and perform a sterile filtration. The
sterile LB glycerol is aliquoted into sterile tubes (max. 10 ml per tube) and stored at
4 C.
For glycerol stocks, this solution is added 1:1 to the bacterial solution (i.e. 0.5 ml
LB glycerol and 0.5 ml bacteria). Ideally the bacteria are flash-frozen (in dry
ice/methanol bath) and then they are stored at -70 C.
223
CDNA SYNTHESIS FROM mRNA AND PCR AMPLIFICATION

1. Material
Solutions :
Saturated phenol with 10 mM Tris-HCl pH 7.5, 1mM EDTA (Merck Nr.206)
Chloroform/isoamylalcohol (24:1) (Merck Nr. 2445 and Merck Nr.979)
Na acetate 3M pH 5.2
EtOH 100 %
Starting mix :
Stock concentration
Final concentration
KCl 2M
DTT 0.1M
Tris-HCL pH 8.6 0.5M
MgCl2 1M
dNTP 25mM
75mM
1mM
50mM
15mM
1mM
For 20g RNA :

400ng oligo dT
50U RNase inhibitor (Boehringer) (=RNasin)
50U RT AMV (Boehringer)
1 l RNase/DNase free (Boehringer)
For PCR :
Primers 50 pmol/l
dNTP : mix of 25 mM each
Taq polymerase 5U/l (Perkins Elmer Cetus, Nr. Nr801-0060)
Parafin oil
2. Protocol
2.1 Synthesis of single strand cDNA by reverse transcription of mRNA
Synthesis Mix :
Final concentration
Stock concentration
75mM KCL
1 mM DTT
15mM MgCl2
400ng olgo dT
50U RNasin
1mM dNTP
20 g RNA
50U RT AMV
H2O
KCl 2M
DTT 0.1M
Tris-HCl 0.5M
MgCl2 1M
oligo dT 1g/l
RNasin 40U
dNTP 25mM
1.9 l
0.5 l
5l
0.75 l
0.4 l
1.25 l
2 l
RT AMV 24U/l
2 l
to 50 l
224
Volume/50l
Incubate 3 minutes at 65 C then 1 hour at 37 C.

Boil 5 minutes.
Add 1 l RNase and digest for 30 minutes at 37 C.
Extract with 1 volume phenol/chloroforme-isoamylalcool (50:50) (facultativ)
Precipitate with 4 l Na acetate 3M and 2volumes of EtOH 100 % during 1 hour at 4C.
Spin 15 minutes at 13 000 RPM in cold room.
Wash with EtOH 70 % (facultativ) and dry the pellet in the dessicator.
Ressuspend the pellet in 50 l of H2O.
2.2 PCR reaction
1 l dNTP
1 l each primers
1 l cDNA
10 l 10x taq Buffer
0.5 l Taq
H2O to 100 l
100 l oil
PCR conditions :
1' at 95 C
1' at 55 C
1' at 72 C
30 cycles
225
MAKING COMPETENT CELLS

Grow bacteria (XL1B, DE3, DH5) in small-scale culture (about 10ml) overnight at
37C, 245 rpm. Next day, add these 10 ml to a final volume of 1l LB-medium. Keep
shaking until OD600 is 0.5 to 0.7.
Cool on ice for 15-30 min. Spin using 250 or 500ml ml sterile plastic bottles 15
min. at 4C (Kontron rotor A6.14 at 4500 rpm or A6.9 at 4200 rpm).
Resuspend in 1l sterile dd water at 4C; spin at same speed. Resuspend in 500 ml
sterile dd water at 4C; spin again the same way. Resuspend in 8-10 ml of sterile 10%
glycerol. Transfer to Greiner(Falcon) tube. Spin 15 min, 4C at 5500 rpm in Kontron rotor
A 8.24.
Resuspend in 1.4 ml of sterile 10% glycerol (conc. about 3x10 10 cells per ml). Precool and label lid of about 40 sterile Eppendorf tubes. Pipette 60-65 ul into each tube. Flash
freeze in dry-ice/methanol bath. Store at -70C.
Comments:
AQ, 9 Nov. 1994
226
ALKALINE MAXI DNA PREP/CSCL2-GRADIENT

This protocol leads to large quantities of DNA (up to 200 g) suitable for sequencing. The
protocol is basically Andrew Quest's protocol of the 2/11/94, with minor changes.
1.
Grow 200 ml bacteria (eg XL1B) in L-broth with antibiotica selection
(ampicillin: 50 g/ml; kanamycin: 25 g/ml)
to stationary phase (12-16 h).
2. Transfer to 250 ml bottle, balance bottles, and centrifuge at 6000 x g (A6.14, 6000
rpm) for 10 min.
3. Decant supernatant (cells in supernatant have to be destroyed by adding bleach
before pouring into sink!) and drain last drops of LB by placing bottles upside down
for 3 min. on paper.
4. Add 5 ml of solution Birnboim I. Resuspend bacteria pellet completely with a 1 ml
Socorex pipet. Add 1 ml of fresh lysozyme solution (10 mg/ml lysozyme). Incubate
10 min. on ice.
5. Add 10 ml of solution Birnboim II and mix gently but immediately by inverting the
tube 4-6 times. Incubate on ice 10 min.
6. Add 10 ml of solution Birnboim III, mix carefully by inverting the tube 4-6 times.
Incubate on ice for 10 min. (up to 3 hrs).
7. Spin at 4 C, 20000 x g (A6.14 rotor, 11000 rpm) for 20 min. (up to 1 hr).
8. Pour supernatant (approx. 25 ml) into new 250 ml bottle, add 9 ml of 8 M
ammonium acetate and 70 ml 100% ethanol. Mix well. Let sit at -20 C for at least
30 min. (evt. overnight).
9. Spin at 4 C, 11000 x g (A6.14 rotor, 8500 rpm). Decant supernatant, let drain
uspide down on paper for a few minutes.
10. Resuspend pellet in 3 ml TE solution, transfer to 15 ml Falcon tube (Falcon 2059,
polypropylene) and measure volume transferred. Wash big bottle with volume
missing to yield precisely 4 ml final volume.
11. Add 4.5 g CsCl2, dissolve by inverting the tube repeatedly, warm to 37 C if
necessary to enhance solubility.
12. Add 0.45 ml ethidium bromide (10 mg/ml in H 2O). Be very careful with this
mutagenic compound!!
13. Centrifuge for 15 min. in clinical centrifuge at 4000 rpm. Pipet supernatant into 6 ml
Quick-seal polycarbonate tube (Kontron #9091 90386).
14. Balance two tubes against each other using CsCl 2 solution (1g/ml H2O) if necessary.
Both tubes should be filled up to the neck - otherwise they collapse. The tubes have
227
to balanced with the corresponding plugs (plastic plug and aluminium cap). Insert
plastic plug.
15. To seal tubes with aluminium cap use special device in hallway (centrifuges). Insert
tubes into special adaptor and fix it well. Then smoothly press down handle until it
clicks. Check whether tube is tightly closed!
16. Insert tubes into TV865 rotor and close occupied tube holes with special lids (use
special tool, do not tighten to hard - you have to open again!).
17. Place rotor into Kontron ultracentrifuge (Centrikon T-2070). Close door and
program: 50000 rpm, 20 C, at least 15 h, vertical on.
18. Next day stop machine, remove rotor, loosen caps very slowly and remove tubes
from rotor carefully. Pull DNA band under UV light. Minimize exposure to UV.
Poke small gauce needle into top of Ultracrimp tube to allow pressure equilibration.
Pull supercoiled DNA band roughly in the middle with 10 ml syringe and large
gauge needle, so as not to shear the DNA. Plasmid DNA should be recovered in
about 1 ml.
19.
Transfer the plasmid DNA into 15 ml Falcon tube. Add up to 4 ml volume with
sterile water. Extract ethidium bromide 3-5 time with water saturated isoamyl
alcohol, until organic phase is colorless.
Maybe spin in clinical centrifuge to enhance phase separation.
20. Add 8 ml ethanol to water phase and precipitate DNA for at least 30 min. at -20 C.
23. Centrifuge for 20 min. at 4 C 12000 x g (A8.14, 10000 rpm). Wash pellet with icecold 70% ethanol, briefly spin and leave air-dry the pellet for 5 to 15 min. For
transfectable DNA, resuspend DNA in 4 ml TE and start again at step 13 (do the
gradient twice).
24. Dissolve pellet in 400 l TE and measure absorption at 260 nm and at 280 nm
(A260=1 -> 50 g/ml DNA). For good quality of DNA A 260/A280 should be between
1.7 and 2.
Plasmid name
A260
A280
228
A260/A280
conc (g/ml)
Solutions
Birnboim I
Birnboim II
Birnboim III
50 mM glucose (9g/l), 10 mM EDTA, 25 mM Tris (pH=8.0);

100 g/ml RNase A
0.2 M NaOH, 1% SDS
3 M KOAc, 0.6 M HOAc
TE
10 mM EDTA, 25 mM Tris (pH=7.4)
229
TRANSIENT TRANSFECTION IN COS

Solutions
DMEM high glucose (Gibco BRL, Nr. 04101966M)
DMEM high glucose/Hepes 10 mM (Sigma, Nr. H-3375)
DMEM high glucose/Hepes 10 mM/5% FCS (= complete medium)
100 mM chloroquine (Diphosphate salt, Sigma, Nr C-6628) in PBS
1000X stock solution, freshly made and filtered through a 0.2 m EinmalFilterhalter (Schleicher & Schuel FP 030/3) for sterilisation
DEAE-DEXTRAN (Chloride form, Sigma Nr. D-9885) in PBS
100X stock solution 40 mg/ml, filtered as before and stored at 4 oC)
10% DMSO (Fluka, Nr 41640) in PBS
Versene (Gibco BRL, Nr. 1540-033)
Method
1. Plate COS cells the day before transfection
- small flask T25 : 1-2 * 106 cells with 5 ml DMEM complete medium
- big flask T75 : 3-4 * 106 cells with 10 ml DMEM complete medium
2. Wash twice the cells with DMEM/Hepes
3. Add 5 ml DMEM/Hepes containing 100 M chloroquine, 400 g/ml DEAE-DEXTRAN
+ DNA (in T25 1g and in T75 2-4 g)
4. Incubate at 37 oC for 2-4 hours
5. Remove medium by aspiration
6. Add 5 ml 10% DMSO in PBS
7. Incubate at RT for 2 minutes
8. Remove medium by aspiration
9. Add 10 ml of DMEM complete medium
10. Incubate at 37 oC for 40-72 hours
For FACS or cell surface proteins experiments, detache the cells from the flask by treating
them for 10 minutes with 2 ml Versene in the incubator at 37 C. Box the flask and suspend
the detached cells in PBS or what ever solution needed for current experiments.
230
PREPARING AND RUNNING SEQUENCING GELS

This protocol is suitable for the following equipment: high potency power supply (eg
GIBCO-BRL ST3002, up to 250 W), running support GIBCO-BRL S2 (approx. 32 x 39 cm
gel).
Choose plates
1. Smaller and bigger plate have to be equally broad.
2. Check whether shark tooth comb sticks easily between the plates. Note slanted edges on
small plate to facilitate comb insertion.
Clean plates
1. Clean with wetrok reocid (or similar non-abrasive detergent).
2. Clean well with normal water (no more foam).
3. Rinse with deionized water.
4. Rinse with 100% ethanol. Dry well.
5. Silanize the plate marked "rep" twice by adding pasteur pipette full of silanizing
solution and distributing evenly over plate surface with paper tissue. Let dry.
6. Wash this plate with deionized water. Dry well.
Prepare plates
1. Put spacers on larger plate and then smaller plate on both. Be sure that
spacer fits
well.
2. Seal plates with Scotch Masking tape (about 5 cm) on both sides, additionally
on
bottom.
Prepare gel solution
1. Mix 46 g urea, 10 ml 10xTBE, fill up with nanopure water to about 80
ml.
To
facilitate dissolving warm to 37 C and swirl gently.

2. Add 15 ml acrylamide solution (40%, 19:1 acrylamide:bisacrylamide).
3. Adjust volume to 100 ml with nanopure water.
4. Add 250 l 10% APS and 90 l TEMED. Mix shortly and pour gel.
5. Insert shark tooth comb with flat surface facing gel.
6. Add clamps to top of gel to create a tight seal between glass plates and
shark
tooth comb.
7. Let polymerize for 2 to 20 h.
Preparation for electrophoresis
1. Pour some nanopure on place where comb is insert and leave there for 1 to 5 min.
Comb can be removed more easily.
2. Prepare about 1 l of 1xTBE. Prepare electrophoresis apparatus.
231
3. Remove gently the comb and Scotch tape.

4. Insert gel into apparatus and preheat 0.5 - 1 h at 2000-2500 V, P=65 W.
5. Rinse well the gel surface.
6. Insert shark teeth comb with teeth towards gel. Comb teeth should insert
into
gel
about 1mm to prevent sample spreading.

7. Heat samples for 5 min. at 75 - 80 C, load about 3 l.
8. After about 1.5 h the darker color front reaches the lower buffer chamber.
After electrophoresis
1. Smoothly separe plates. Gel should stick to smaller plate.
2. Put gel very carefully into a solution of 10% methanol, 10% acetic acid (2l should be
enough) and leave there for about 0.5 h.
3. Prepare chromatography paper of size 39 x 32 cm (Whatmann 5mm
best, S&S GB 002 is ok).
4. Recuperate fixation solution. You can reuse about ten times.
5. Put paper on gel, so that it fits well. Never try to remove paper from gel!
6. Roll several times with pipette over paper, applying moderate pressure.
7. Dry gel for about 2 hrs at 80C (Fasel's big dryer).
232
works
SAMPLE PREPARATION FOR DSDNA SEQUENCING

Biotechniques 6(5), 460-469, 1988. F. Toneguzzo, S. Glynn, E. Levi, S. Mjolsness, A.
Hayday. Use of chemically modified T7 DNA polymerase for manual and automated
sequencing of supercoiled DNA.
1. 2-4 ug supercoiled DNA & 10-50 ng primer (1: 10 molar ratio as a minimum). Call this
volume "y" = .......... ul
233D
Primer
H2O
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
2. Add equal volume (i.e. vol "y") of 0.4 M NaOH/0.4 M EDTA (pH=8.0). Make this fresh
(eg 800 ul 0.5 M EDTA, 40 ul 10 M NaOH, 160 ul H2O).
3. Heat 85o C for 5 min and ice immediately.
4. Add vol "y" of 7.5 M ammonium acetate and 9 vol "y" of 100% EtOH.
5. Keep at least 20 min. at -20o C. Spin in microfuge 15 min. in cold.
6. Wash pellet with 100% EtOH and dry in desiccator (You can store this way in -20 o C for
up to two months).
7. Resuspend DNA in 7.5 ul of mix made as 6 ul H 2O and 1.5 l 5x Sequenase buffer (eg 9
reactions: 14 ul buffer, 56 ul H2O).
8. Incubate 37o C for 15 min.
9. Add
1 ul 0.1 M DTT
2 ul labeling mix (standard 1:5)
1 ul labeled nucleotide
2 ul Sequenase (1:8 diluted in cold TE)
10. Incubate at room temperature for 1 to 10 min. Probably 2 min. is fine.

11. Add 3 ul labeling reaction to each warmed tube containing 2.5 ul termination mix for G,
A, T and C.
12. Incubate at 37o C for 15 min.
13. Add 4 ul stop buffer.
14. Heat 75o C for 3 min prior to loading.
15. Load 3 l per slot on gel (about 45 teeth).
233
GEL ISOLATION OF RESTRICTION DIGESTED PCR-DNA

DNA samples were run out on agarose gels after restriction digestion. The agarose
bands containing the appropriate DNA band are cut out and DNA must be isolated. This is
done using the Geneclean II Kit (BIO 101 Inc.).
To the agarose add 3X the volume of NaI Stock. Warm up to about 55C until the
agarose is completely dissolved. Then let cool down to room temperature. Add
GLASSMILK (mix well, use 1 ul per ug DNA; generally about 5 ul) to the tube and mix
well. Leave standing 5-10 min. and mix every few minutes. Then spin briefly for 5 sec. to
collect pellet. Remove sup. (do not throw away) and wash the GLASSMILK pellet with
three times with about 500-800 ul of NEW WASH. Discard sup. each time. At the end elute
bound DNA at 55C, first with 15 ul TE, then with a second time with 18 ul TE. Collect
both sups. (do not discard matrix yet) and analyze one tenth, i.e. 3-4 ul on a gel to make
sure DNA was recovered and to get an idea how much to use in the subsequent ligation
step. With DNA500 bp, recovery should be 80-90%; for smaller fragments it will be lower.
Only throw away matrix and initial NaI-agarose sup. when you know that the DNA was
recovered.
Analysis on a 1.5-2% gel:
Comments:
AQ, 8. sep. 1994
234
LIGATION PROTOCOL FOR DOUBLE DIGESTED PCR-DNA & PGEX2T
DNA samples must now be ligated into pGEX2T or other plasmids like pGEXKT,
pCMV4his etc. The combination of restriction sites exposed by digestion (choice of BamHI
or BglII with KpnI or ECoRI) should match the sticky ends of the double-digested vector
the PCR-DNA will be inserted into.
SAMPLE
INSERT
VECTOR
MOLAR RATIO
DNA RATIO
Code size(bp)
size(bp)
(insert:vector)(insert:vector)
1/
2/
3/
4/
5/
6/
7/
8/
9/
10/
11/
12/
Ligation conditions:
1
2
3
4
5
6
7
8
9
10
11
H2O
vector
insert
buffer(10x)
ligase
Leave at room temperature for 4-6 hrs or o/n at 4C in styrofoam box. Ethanol
precipitate to remove ligation buffer.
EtOH ppt: to 20 ul volume add 1ul tRNA (2ug/ml), 3 ul 3M sodium acetate, 40 ul 100%
ethanol, mix well leave 15-30 min. at -20C, spin in Eppendorf centrifuge at 13000 rpm for
20-30 min. at 4C, remove supernatant and dry pellet in vacuum desiccator.
Resuspend in 5-10 ul sterile dd H2O.
AQ, 8. sep. 1994
235
PLASMID MINI BOILING PREP

This protocol yields DNA for restriction analysis and as a template for PCR. With
an additional phenol:chloroform extraction the DNA is also suitable for sequencing.
1- Grow 3 ml o/n culture in LB plus ampicillin (100 ug/ml)
2- Pellet 1.5 ml in Eppendorf tube - if necessary pellet rest as
well (this may be
necessary for slow growing strains like XL1B)

3- Resuspend pellet in STET (8% sucrose, 5% Triton X-100, 50 mM
8.0, 50 mM EDTA, 0.5 mg/ml lysozyme) . All components
prepared and stored indefinitely at 4C
Tris
pH
except lysozyme can be
(mix 2.2 ml 2M sucrose, 0.75 ml Tris pH 8.0,
1.5 ml EDTA, 7.5 ml 10% TX100 or NP40, add dd water to 15 ml volume). The lysozyme
is made as a 50 mg/ml stock, stored in small aliquots frozen
at -20C which are
only thawed once, and added just prior to usage.

4- Place tube in boiling water bath for 30 seconds.
5- Add 50 ul 500 mM EDTA and mix well. White gel should occupy
volume. Spin for 30 min. to obtain a good pellet, which can
be
small
removed
with
toothpick or a yellow pipette tip; discard.

6- To the remaining solution add 0.2 ul of RNAse free DNAse
Mannheim #1119915, 500 ug/ml). Treat for 1 hour at
(Boehringer
37C. This step removes RNA
which is so prominent that it would be hard to see small inserts when cut out of plasmid
with
restriction enzymes. This treatment also facilitates subsequent
Then add 110 ul of isopropanol and spin

(DNA) carefully with
sequencing steps.
immediately for 15 minutes. Wash pellet
70% ethanol.
7- Resuspend DNA in 20 ul TE. DNA is adequate for restriction analysis or as a

PCR template.
Gel (1.5-2%) analysis of plasmid DNA by PCR:
To obtain sequencing grade DNA continue as indicated below:

Extract with an equal volume of phenol:chloroform (1:1) 2 times and
once
with
chloroform.
Add an equal volume of 7.5M ammonium acetate and precipitate with 2.5 volumes
of ethanol. Leave on ice for 15 min. and spin at
4C for 20 min.
Rinse carefully with 70% ethanol; dry pellets under vacuum.

236
Resuspend in 15 ul TE and use 5 ul for sequencing (about 2 ug).
Comments
AQ, 13. sep. 1994
237
MINIPREPARATION OF PLASMID DNA BY ALKALINE LYSIS

(Rosa; 25.9.98)
Solutions:
Birnboim I
9 g/l Glucose
25 mM Tris-HCl (pH=8)
10 mM Na2EDTA (pH=7.4)
Birnboim II
0.2 M NaOH
1%
SDS
Birnboim III
3.0 M KAc (299 g/l)

0.6 M HAC (34.3 ml/l)
Take 1.5-3 ml of overnight E. coli culture. Pellet bacteria, remove as much supernatant
as possible (last bit with yellow tip) and resuspend pellet in 200 l Birnboim I by
pipetting up and down with yellow tip.
Add 200 l of BirnboimB II, mix thoroughly by inverting the tube several times until
solution becomes clear. Incubate at room temperature for 5 Min.
Add 100 l of chloroform and neutralize by adding 200 l of Birnboim III, mix well by
inverting the tube.
Precipitate during 10 Min. at 13'000 rpm in a minifuge at RT.
Take away 500 l of supernatant that contains DNA (if necessary using with drawn-out
pasteur pipette) and put into new tube.
Precipitate DNA by addition of. 350 l of isopropanol. Pellet DNA by spinning 5 min.
at 13'000 rpm at room temp.
Wash pellet with 600 l cold (20C) 70% ethanol, spin for 3 min at RT 13'000 rpm.
Dry pellet and resuspend pellet in 20 l of water.
Add about 1 l DNAse-free RNAse (Boehringer Mannheim; Cat.Nr.1119 915)(1:5

diluted in water, 50 ng RNase per sample) and incubate for at least 20 minutes at room
temperature.
Expect about 2.5 g DNA per ml bacteria culture.
238
NORTHERN ANALYSIS
Best way is to use RNA ribo probes. These are generated using a cDNA template
inserted into for instance Bluescript, which allows T7 or T3 dependent transcription or
entire sequence into RNA. Depending upon whether you want sense or anti-sense RNA one
or the other RNA polymerase promoter is used. At the same time the RNA is labeled with
P32. This requires preferably a full-length cDNA containing plasmid. NF has protocols.
Next best shot, especially if cDNA is not available, is to use PCR primers to
specifically PCR out the cDNA of interest starting from mRNA. This is more time
consuming, but will eventually lead to the same results.
Apparently worst option is to use oligos as probes. First they have to be at least 2530 nucleotides long for specificity. Then they are only endlabelled, so the label content is
lower than with riboprobes. Also there is almost always a problem of background with 28S
and 18S RNA.
In terms of doing the experiment, Marga knows how to do everything except the
actual probing. NF has all the protocols.
Precaution: after running a gel cut a piece at the end and stain with ethidium bromide to
know where 28S and 18S RNA migrate. Mark positions on blot after Northern Transfer.
After prehybridization, blots may be stored away until usage. Probe first with marker for
RNA presence, i.e. actin, tubulin, IL2 (all available with NF). Then strip and reprobe with
the probe of interest.
239
PCR WITHOUT HOT START

Here at the BIL there are no ampliwax pellets that would allow a hot start. For longrange PCR this would be very desirable. For the short constructs we wish to generate most
of the time (300 - 500 bp), it probably does not matter. The PCR program I use here simply
starts with a very rapid heating step up to 95 C (within a minute or two), before the actual
cycling routine begins.
To do PCR make master mix without Taq polymerase, primers or template DNA
with amounts indicated for one reaction as follows:
10 ul PCR buffer 10x (with magnesium)
10 ul 1 mM dNTP mix (made from 20 mM Stocks)
80 ul dd sterile water
To each sterile PCR tube add 96 ul master mix, then
1 ul sense primer (500 ng/ul)
1 ul anti-sense primer (500 ng/ul)
1 ul template DNA (100 ng/ul)
1 ul Taq polymerase diluted 1:1 in master mix (2.5 units per tube)
Overlay mix in each tube with mineral oil (Sigma); make sure there is enough to cover the
entire liquid surface, otherwise your volume changes during the PCR reaction. This can
effect the result adversely.
Program thermocycler to stay 1 min at 95C, to aneal at 55 C for 1 min (temp.
depends on primers; as a rule of thumb 10 C below melting temp of primer/DNA), to
extend at 72 C for 2 min (time depends on length of fragment desired; increase for longer
constructs), to melt again at 95C for 1 min etc. This is repeated 30 - 40 times after which
the thermocycler is programed to cool to 4 C and stay there until you turn the machine off
(equivalent to pgm 44)
Precautions: As soon as you are dealing with the primers use either flow-restricted tips (e.g.
ART tips from Continental Lab Products) or Eppendorf pipetteman and clean cylinder
every time with ethanol when changing primers.
PCR Reaction Series:
date
Goal:
NB. When you are making new constructs using fresh template DNA preps, new batches of
primers etc. always include a positive and a negative control for the PCR reaction.
240
Sample Nr.
Construct
Name
Primers
Fragments (bp)
SN(5)/AT(3) predicted/gel
1
2
3
4
5
6
7
8
Results of PCR reaction: Analyze 10 ul of each PCR reaction on a 2% agarose gel together
with DNA VI markers (1ug) from Boehringer Mannheim. Photograph under UV: aperture
8, 0.25 exposure, no filter
sample number
DNA marker
241
PCR TO CHECK PLASMID DNA FOR INSERTS

This protocol is similar to that used for the initial PCR reaction to obtain segments
of interest except that PCR volume and the amount of Taq employed are reduced.
Plasmid Miniprep DNA code Nr:
(3 preps per insert should be enough)
1/
2/
3/
4/
5/
6/
7/
8/
9/
10/
(primers)
Follow protocol, except add 50 ul 500 mM EDTA after boiling samples. Vortex
well. Particulate matter should not be jelly-like. Resuspend DNA in 20 ul TE; use 2 ul for
PCR.
Per 24 samples, plus a positive control, make master mix for 25 samples, w/o Taq
polymerase, primers or template. Per tube 50 ul
Tot. volume
containing
PCR buff. 10x

1 mM dNTP mix
dd H2O
Taq polymerase
(N.B. The volume of Taq is half that normally employed).
1.25 ml
125 ul
125 ul
1.0 ml
6.5 ul
To each tube add
48 ul mix
0.5ul 3 primer
0.5ul 5 primer
1.0ul mini prep DNA
Overlay mix in each tube with mineral oil (Sigma); run PCR using standard program.
Gel analysis using 20 ul of PCR product in comparison with 1ul miniprep DNA on 250
ml,1.5% agarose gel. DNA VI markers (1ug) from Boehringer Mannheim. Photograph
under UV: aperture 8, 0.25 exposure, no filter
sample number
DNA marker
positives:
predicted size/actual size
Grow overnight cultures of all positives for glycerol stocks which are made by mixing
242
culture1:1 with LB containing 30% glycerol, and freezing at -70C.

Characterize constructs, starting with the one highlighted, by ds DNA sequencing off pGEX
and expression of the respective fusion protein if sequencing looks good.
243
ISOLATION OF PCR FRAGMENTS FOR LIGATION

The primers have been designed so that the PCR products contain at least one set of
restriction
Sample Nr.
Construct
Name
Primers
Fragments (bp)
SN(5)/AT(3) predicted/gel
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Results of PCR reaction: Analyze 10 ul of each PCR reaction on a 2% agarose gel together
with DNA VI markers (1ug) from Boehringer Mannheim. Photograph under UV: aperture
8, 0.25 exposure, no filter
sample number
DNA marker
244
ALKALINE PHOSPHATASE TREATMENT OF DNA

Protocol:
Use 1 unit of phosphatase (Boehringer Mannheim; Cat.Nr.1097075) per 60 l
volume of double-cut DNA (3-5 g DNA) eluted from Quiagen matrix in TE
Incubate for 2 hours at 37C in waterbath
Stop reaction by adding EGTA and heating to 65C for 10 min re-isolate DNA
using Quiagen kit and 10 l matrix. Elute DNA in 2 times 20l TE.
With 50 ng DNA background should only be 10-20 colonies even if vector was
only digested once with two different restriction enzymes.
Alternative:
double digest and isolate plasmid DNA from agarose gels two times consecutively.
Background should also not be higher than 10-20 colonies per 50 ng.
NB:
Using phosphatase treatement after a single round of digestion with 2 different
restriction enzymes generally works more reliably. However, upon plating background is
apparent as a large number of very small colonies. Only large colonies contain inserts,
generally with very high efficiency.
245
PROTOCOL TO GENERATE POINT MUTATIONS AND AT THE SAME TIME

RESTRICTION SITES
(according to Pascal Schneider May, 2001)
a) write the mutated amino acid sequence you want with a few aa on each side, then write
below the corresponding nucleotid sequence.
b) under the nucleotide sequence, write all codons which do not change the amino acids
c) look for restriction site (by eye) at or close to the mutation. For this purpose, start at the
center of the site, which is either CG, GC, TA or AT, using all the possible sequences that
were written down. Each time you find these sequences, look at the surrounding aa. If it is
still a palindrome (eg. TGCA), look at the next two bases and if they still are palindromic,
you have a restriction site (eg ATGCAT).
d) Make a copy of the Roche/Boehringer catalogue where they have a nice summary table
of RE and sites and names. Find your sequence, see if there is an enzyme corresponding to
the sequence, and decide whether it is one which is suitable.
e) Check that the site you introduce will allow you to distinguish wt from mutant (e.g. the
same siet is not present close to the one you generate) and that it won't prevent your cloning
strategy.
f) choose your primers so that:
1.- there are 15 or more bp 3' of the last mutated base
2.- primer ends (if possible) with a G or a C at 3'
3.- forward primer starts in 5' just after a T (will allow to use Taq which
leaves a 3' A overhang, if Pwo doesn't work)
4.- reverse primer starts just after an A (if Taq is used)
5.- overlap between the two primers is 15 bp or more
g) order and perform experiment.
246
PROTEINASE K TREATMENT OF PCR FRAGMENTS

The PCR mixture contains the anticipated DNA fragments, template DNA,
nucleotides and Taq polymerase. If Taq and nucleotides are still present when sticky ends
are being created, for instance using restriction enzymes, Taq can start adding nucleotides
at
random to these ends. Addition of
a single nucleotide is sufficient to
prevent
subsequent ligation. To avoid this, the PCR products are treated with proteinase K
immediately after being generated. This improves dramatically subsequent ligation
efficiency (ref. Bennett & Molenaar (1994) Biotechniques 16, pp32-37). Problematic with
this step may be the recovery of the DNA by ethanol precipitation.
After analysis on an agarose gel roughly 90 ul of PCR product remain. Remove this
volume from each tube and ethanol precipitate by adding 90 ul of 8M ammonium acetate
and 360 ul 100% ethanol; mix and leave at -20C over night. Next day, recover pellet by
centrifugation in Eppendorf centrifuge for 20 min. at 4C. Remove supernatant very
carefully, dry pellet in dessicator under vacuum. Resuspend in 87.5 ul Tris/EDTA pH 8.
Add 10 ul Proteinase K stock in TE at 500 ug/ml and 2.5 ul 20% SDS. Incubate 30 min. at
37C and then 10 min. at 68C. Ethanol precipitate again by adding 100 ul 8M ammonium
acetate and 400 ul ethanol; mix and leave at -20C at least an hour or over night. Recover
pellet by centrifugation in Eppendorf centrifuge for 20 min. at 4C. Remove supernatant
very carefully, dry pellet in dessicator under vacuum. Resuspend pellet in 45 ul TE.
Comments:
AQ, 2. sep. 1994
247
RESTRICTION DIGEST OF PROTEINASE K TREATED DNA

DNA samples must now be digested with appropriate restriction enzymes to allow
ligation into pGEX2T or other plasmids like pGEXKT, pCMV4his etc. The combination of
restriction sites in the initial PCR primers should have been chosen such that no internal
restriction sites are present in the PCR products.
SAMPLE
Code/Name
RESTRICTION
enzyme comb.
CONDITIONS
(volume ul)
PCR DNA
Buffer 10x
dd H2O
RE I
RE II
PCR DNA
Buffer 10x
dd H2O
RE III
RE IV
Digest with each restriction enzyme for at least an hour at 37C. Then run samples out on
1.5-2% agarose gel (250 ml, 3x26 slots). Isolate double cut bands and prepare for genecleanT M isolation of gel
Digest Results:
Code/Name
Agarose
Na I Stock
wt. (g) vol. (ml)
photo
1/
2/
3/
4/
5/
6/
7/
8/
9/
10/
11/
12/
Comments:
AQ, 7. sep. 1994
248
RESTRICTION DIGEST OF PLASMID DNA

Objective:
SAMPLE
Code/Name
RESTRICTION
enzyme comb.
CONDITIONS
(volume ul)
DNA
Buffer 10x
dd H2O
RE I
RE II
DNA
Buffer 10x
dd H2O
RE III
RE IV
0.8-2% agarose gel (250 ml, 3x26 slots). Isolate double cut bands and prepare for genecleanT M isolation of fragment if desired.
Digest Results:
Code/Name
Agarose
Na I Stock
wt. (g) vol. (ml)
photo
1/
2/
3/
4/
5/
6/
7/
8/
9/
10/
11/
12/
Comments:
AQ, 2 Nov. 1994
249
PREPARATION OF mRNA FROM CULTURE CELLS

1. Use confluent culture on large culture dish (adherent cells) or appropriate
amount of
ConA blasts (about 107 cells)

2. Discard medium, wash cell layer briefly with cold PBS
3. Detach cells using rubber policeman and transfer to 15ml Greiner tube. Spin 5 min. at
2500 rpm (Kontron A 8.24 rotor), 4C
4. Resuspend pellet and wash 1x with cold PBS. Spin again 5 min. at 2500rpm and remove
supernatant
5. Add 2ml of cold RNA lysis buffer. Vortex 10sec. Add 50ul VRC (RNAse inhibitor). Spin
5 min. at 4C, 7000rpm. Transfer supernatant to 50ml Falcon tube, add 2ml PK buffer (RT)
and 40ul Proteinase K solution. Incubate 30-60 min. at 37C
6. Add 2ml phenol and 2ml chloroform, then spin 5min. at 2000rpm, RT. Recover aqueous
phase and repeat extraction 2x.
7. Transfer aqueous phase to a Greiner tube and add 8ml ethanol and 266ul 3M NaAc.
Precipitate for a minimum of 3 hours or maximum o/n at -20C.
8. Spin 30min. at 7000rpm, 4C. Wash pellet again once with cold ethanol
and
resuspend in 100-200ul (volume as small as possible) ddH2O

9. Measure OD 260nm, calclate RNA conc. with 1 unit at OD260 = 40 ug/ml
Check quality of RNA preparation by running an 0.8% agarose gel. 28S and 18S RNA
bands should be clearly visible.
NB. All manipulations are done on ice wearing gloves.
Use perfectly sterile material and preferably labware set aside for work with RNA
only.
SOLUTIONS
Lysis Buffer:
mix 28 ml 5M NaCl, 0.15 ml 1M MgCl2, 1ml 1M Tris-HCl pH 8.6,

2ml 25% NP40, and fill up to 100ml final volume with ddH2O
2x PK Buffer:
20ml 1M Tris-HCl pH7.5, 5ml 0.5M EDTA, 6ml 5M NaCl, 20ml

250
10% SDS, and fill up to 100 ml final volume with ddH2O

PBS 10x:
40g NaCl, 7.12g Na2HPO4.2H2O, 1g KCl, 1g KH2PO4,, and fill up

to 500ml with ddH2O
VRC 200mM RNAse inhibitor: complex of Vanadium IV oxide sulfate pentahydrate (Fluka
#94730)/ Adenosine (Merck #862). Suspend 1.3g of VOSO4 in 4ml ddH2O by warming
solution. Prepare 250mM adenosine by dissolving 1.67g adenosine (Mw 267) in 24ml
warm ddH2O. Add dropwise warm VOSO4 to adenosine solution and mix well. The colour
changes to green-black and the pH drops to about 2.5. If the reagent is added too quickly, a
black ppt. will form. After adding adenosine, immediately add a few drops of 10N NaOH
to adjust the pH to about 6, add 3ml of water to a final volume of 30ml and make final pH
adjustment to pH7 with 1N NaOH. Store the 200mM VRC solution at -70C
Proteinase K 20 mg/ml (Boehringer Mannheim #161519)
Phenol saturated with Tris-HCl 10mM, EDTA 1mM
Chloroform:Isoamylalcohol 24:1
Ethanol 100%
NaAc 3M, pH adjusted to 5.2 with glacial acetic acid
ddH2O, sterile double destilled water
Comments
AQ, 2 Nov. 1994
251
TRANSFORMATION PROTOCOL FOR LIGATED DNA

Ligated DNA samples must now be transformed into an appropriate E . coli strain.
We use mainly XL1B and BL21 for subsequent expression of GST fusion proteins.
Competent cells take up the DNA by electroporation.
Procedure:
The electrode holder of the electroporator and the cuvettes are put on ice at least an hour
before transformation. DNA from ethanol precipitation resuspended in water (max. 10 ul) is
mixed with an aliquot (40 - 50 ul) of competent cells that have just been thawed and is
incubated briefly on ice (1 - 5 min.). Then cells with DNA are placed in cold electroporator
cuvette, making sure that cell suspension is at the bottom of the cuvette. Pulse cells (setting
2.5 V; 400-450 s). When beep tone starts, immediately remove cuvette, add 1 ml aliquot
of SOC medium with glucose, pipette up and down 3x and put in sterile 15 ml conical tube.
Discard cuvette. Cells are incubated in shaker at 37C for 1 hour and then plated 100 - 300
ul on selective agar plates (i.e. + amp.). Colonies that grow should contain plasmid with
insert; efficiency now 80%. Always include a control with double-digested plasmid alone.
Even if your transformants with inserts give lower numbers of colonies than the control,
still check the colonies for inserts anyway by PCR. In many cases I have still been able to
get plasmids with the inserts; efficiency is 30-50%.
colonies per plate:
1
10
volume
plated
transform.
effic. per
ng DNA
Comments
AQ, 9. sep. 1994
252
DNA SEPARATION IN AGAROSE GEL

-PCR products or plasmids cut by restriction enzymes
-Buffer TAE (40mM Tris-acetate pH 8.5, 1mM EDTA)
-1% agarose in TAE buffer
-ethidium bromide at 10mg/ml (this product is very toxic. Use gloves)
-6x DNA sample buffer (10ml of glycerol 87%, 84mg of bromophenol blue, 40ml water,
add a little of Tris 1M until solution is blue)
-DNA standards at 0.1g/l (5l + 5l water + 2l sample buffer)
Method
10) -Prepare the gel in a minigel cuvette (horizontal). Put the 16 well comb and the spacers
besides the electrodes
11) Melt 1% agarose in a microwave or in a wather bath
12) Take 50ml of agarose, Add 2.5 l of ethidium bromide and add all to the cuvette.
Gelification takes aproximately 20 min
13) Add sample buffer to the samples: 2l of 6xSB to the DNA after restriction enzymes
treatment, 6l to DNA of midipreps and 10l to PCR products
14) Withdrawn carefully the comb and spacers. Add buffer TAE (as much as possible,
otherwise agarose melts). Load samples (15l/well)
15) Connect electrodes. Samples migrating to the positive (red) and run the gel at 60V
during 20min (blue bromophenol should run upto the middle of the gel, approx 500bp
16) Trow away buffer on activated charcoal and look at the bands under UV light. If the
fragments are to be extracted, use a lamp of UV short.
253
EXTRACTION AND DNA PURIFICATION FROM AN AGAROSE GEL

DNA samples were run out on agarose gels after restriction digestion. The agarose
bands containing the appropriate DNA band are cut out and DNA must be isolated. This is
done using the QIAEX II Kit (Qiagen, Germany).
The following protocol os for DNA fragments between 100bp-4kb. If DNA is bigger or
smaller refer to manufacturers original protocol.
- To the agarose (weight again empty epp tube) add 3 volumes of QXI Stock (ex. 100mg
agarose- 300l QXI).
- Add 10l of QiaexII beads
- Put in warm bath at 50C for 10 min, mix from time to time (check that color, pH, doesnt
change, if it does, add 10 l of 3M sodium acetate pH5.0 and mix. DNA binding only takes
place at pH 7.5 )
- Eliminate supernatant after a quick spin (15)
- Wash beads with 500l of buffer QXI
- Wash again with 500l of buffer PE (twice)
- Spin and take ALL supernatant carefully. Let beads air-dry for 10-15 min (do let to
overdry)
- At the end elute bound DNA at room temperature for 5 min, first with 20 l sterile water,
then a second time with other 20 l. Collect both sups. in separate sterile tubes (do not
discard matrix yet) and analyze one tenth, i.e. 2 l on a gel to make sure DNA was
recovered and to get an idea how much to use in the subsequent ligation step.
GLYCEROL STOCK PREPARATION
Transfected bacteria could be stored for many years in 20% glycerol. Glycerol protects the
bacteria by avoiding the formation of crystals in the bacteria during the freezing process.
To obtain bacteria out of a glycerol stock, scrape the surface with a sterile inoculating loop
and seed it over a plate or directly in a culture medium containing the appropriate
antibiotics (kanamycin for pCRblunt or ampiciline for pCR3).
254
GLYCEROL STOCKS
should never be thawed
To prepare glycerol stocks you need
-
Sterile eppendorf tubes
Sterile 40% glycerol
Freezer at 70C
Your bacteria culture
27) To prepare the culture inoculate a 10ml of LB + antibiotics with the bateria (or a
colony).
28) Leave it growing overnight at 37C shaking at 200-250rpm.
29) Add 500l of bacteria containing LB to an eppendorf tube containing 500l of 40%
glycerol.
30) Freeze at 70C
255
LIGATION OF TWO DNA FRAGMENTS

Material and solutions:
-Water bath at 16C
-pCRO-blunt at 25ng/l (PS358) linearized with Stul. For pCR3 linearize PS404 with
HindIII and SalI.
-Insert, purified DNA
-10x concentration ligation buffer
-T4 DNA ligase
Method
1.- Pipet the following in sterile eppendorf tubes
1l 10x buffer (thaw only once)
1l of pCRO-blunt (2l for pCR3)
7l PCR product
1l of T4 DNA ligase
2.- Incubate the ligation for at least 1 hr at 16C
3.- Use ligation to transform bacteria
256
MINIPREP OF PLASMID DNA

Material and solutions:
-Shaker for bacteria at 37C
-microcentrifuge
-bacteria colonies from LB-agar plates
-Sterile round bottom tubes of 10ml
-medium LB containing 50g/ml of Kanamycin
-Inoculation sticks
-Buffer P1 (50mM Tris-HCl pH 8.0, 10mM EDTA, 100g/ml RNAse A)
-Buffer P2 (1% SDS in 0.2M NaOH)
-Buffer P3 (3M sodium acetate, pH 5.5)
-Chloroform
-isopropanol
-ethanol 70% at -20C
Method:
-
For each transformation, inoculate 6 colonies in the tubes of 10 ml containing 2.5ml of

LB medium with kanamycin
Leave incubating overnight at 37C with agitation (200-250rpm)
Transfer 1ml of bacteria in 1.5ml tubes
Spin at 13000rpm for 30 sec, throw away all supernatant
Add 200l of buffer P1. Resuspend the bacteria with the pipet
Add 200 l buffer P2. Mix tubes vigourously with the hand
Leave for 4 min at RT
Add 100l of chloroform. Mix
Add 200l buffer P3. Mix
Incubate for 10 min on ice
Spin 3 min at max speed
Transfer 500l of the upper phase without disturbing interface into 1.5ml epp tubes
which already contain 350l of isopropanol, Mix. Recover chloroform in th organic
waste bottle
Spin 3 min max speed. Little white pellet is obtained. Withdraw all supernatant. Spin
again if necessary to get rid of last drop
Wash the pellets with 200l of ethanol 70% at -20C. Eliminate supernatants and dry
pellet 5min at 37C
Resuspend the pellet in 40l of water (sterile)

257
Analyze preparation with restriction enzymes.

Making mThy-1.1-placIOP
1) Get primers for full length mThy-1 and insert a Not1 site at both ends. Protocol 1
2) Amplify by PCR using cDNA from plasmid p826 (from N. Fasel, BIL-CH).
Protocol 2
3) Run in an agarose gel and Purify DNA band. Protocol 3a and b
4) Ligate into pCRblunt. Protocol 4
5) Transfect into competent TOP 10 (or Dh5) cells and select. Protocol 5
6) a. Make a miniprep. Protocol 6a and a glycerol stock. Protocol 6b
b. Analyze with RE to select the right clone. Protocol 7
c. Send plate with the clone for sequencing. Plasmid XXX
7) If OK, subclone in placIOP
a. Cut and purify placIOP vector from plasmid cav-1placIOP with Not1. Protocol 7 and
3a, 3b
fragment size9kb?. (cav-1 is about 580bp)
b. Treat the vector (never the insert) with alkaline phosphatase and do a MOCK
transfection check the background due to autoligation. Protocol 8
c. Cut and purify insert of plasmid XXX. Protocol 7
fragment size.bp
c. ligate both purified fragments (Vector placIOP wuth the Thy-1 insert) Protocol 9
d. transfect TOP 10 cells and select with ampicilin Protocol 5
8) a. Make a miniprep. Protocol 6a and a glycerol stock. Protocol 6b
b. Analyze with RE to select the right clone. Protocol 7
c. Make a Midiprep. Protocol 9
258
PCR AMPLIFICATION
Material and Solutions:
-Thermocycler
-tubes PCR of 0.2-0.5 ml (sterile)
-cDNA or plasmid containing DNA to amplify
-Reaction buffer concentrated 10x (0.2M Tris-HCl pH8.4; 0.5M KCl; 15mM MgCl2)
-dATP, dCTP, dGTP, dTTP all together at 2mM each
-5-3primer at 10M in sterile water (If it come 45nmol, add 450l water to hace a stock at
100M. Dissolve it by heating at 65C for 5 min. Make a dilution of 1 to 10 to have it at
10M. Aliquot and save at -20C
-3-5primer at 10M in sterile water
-Pwo DNA polymerase from Boerhinger-Manheim. It comes at 5U/l in 20l of enzyme
buffer. Dilute with same buffer to 1U/l and use 1l. Try always keeping it at -20C, since
it come in glycerol it will not freeze.
Method
- dilute plasmid or cDNA at a concentration of 0.1g/ml
- prepare the tubes as follows:
1l of plasmid, cDNA (include a positive and a negative control)
5l of 10x PCR Pwo-buffer
5l of 5-3 primer at 10M
5l of 3-5 primer at 10M
5l of dNTP at 2mM each
28l of distilled water (sterile)
1l of Pwo DNA polymerase (add it at the last minute)
-put the tubes in thermocycler (add mineral oil if necessary) and use the pollowing
program:
1.- 3min at 94C

2.- 1min at 94C
3.- 1min at 55C
4.- 1min at 72C
5.- repeat steps 2-4 23 times
6.- 10min at 72C
7.- 4C
-Analyze product in an agarose gel
259
RESTRICTION DIGEST OF PLASMID DNA

-If DNA to be analyzed is not pure, include addition of RNAse. If using pure DNA, 1g of
DNA is enough. If DNA purification is desired add 10-15 g of DNA
-Always add a non-digested control
-Digestion buffer depends on the enzyme. If two enzymes are used and their buffers are
incompatible, it is necessary to precipitate the DNA between each treatment
-Objective:
SAMPLE
Code/Name
RESTRICTION
enzyme comb.
CONDITIONS
a
(volume ul)
DNA
Buffer 10x
dd H2O
RE I
RE II
DNA
Buffer 10x
dd H2O
RE III
RE IV
0.8-2% agarose gel (2x16 slots). Isolate double cut bands and prepare for QiaexII kit
isolation of fragment if desired.
Comments-Results:
260
STRATEGY TO MAKE A POINT MUTATION WITH A RESTRICTION SITE

INSERTED
a) write the mutated amino acid sequence you want with a few aa on each side, then write
below the corresponding nucleotid sequence.
b) under the nucleotide sequence, write all codons which do not change the amino acids
c) look for restriction site (by eye) at or close to the mutation. For this purpose, start at the
center of the site, which is either CG, GC, TA or AT, using all the possible sequences that
were written down. Each time you find these sequences, look at the surrounding aa. If it is
still a palindrome (eg. TGCA), look at the next two bases and if they still are palindromic,
you have a restriction site (eg ATGCAT).
d) Make a copy of the Roche/Boehringer catalogue where they have a nice summary table
of RE and sites and names. Find your sequence, see if there is an enzyme corresponding to
the sequence, and decide whether it is one which is suitable.
e) Check that the site you introduce will allow you to distinguish wt from mutant (e.g. the
same siet is not present close to the one you generate) and that it won't prevent your cloning
strategy.
f) choose your primers so that:
1.- there are 15 or more bp 3' of the last mutated base
2.- primer ends (if possible) with a G or a C at 3'
3.- forward primer starts in 5' just after a T (will allow to use Taq which leaves a 3' A
overhang, if Pwo doesn't work)
4.- reverse primer starts just after an A (if Taq is used)
5.- overlap between the two primers is 15 bp or more
g) order and perform experiment.
261
TRANSFORMATION OF BACTERIA WITH A RECOMBINANT PLASMID

-Water bath at 42C
-Competent Top-10 bacteria (50l aliquots)
-ligation product
-0.5M 2-mercaptoethanol. Sterile
-medium SOC
-LB-agar plates with 50g/ml of kanamycin
- Pasteur pipets with their tip in triangular shape and ethanol
Method
1.- Thaw competent bacteria on ice
2.-Add 2l of 0.5 M 2-mercaptoethanol (mix by swirling, do not pipet bacteria up and
down)
3. Add 2l of the ligation product. Mix as before
4.- Incubate for 30 min on ice
5.- Put tubes at 42C for 45 sec
6.- Incubate for 2 min on ice
7.- Add 450l of SOC medium and incubate at 37C for 1hr under agitation in a horizontal
position. During this time warm up the plates at 37C (open the plate and put lid with
opening to the bottom of the incubator, place the plate with a side on top of the lid, also
inverted)
8.- Take 200l of bacteria and put them in the center of the plate. Spread making a circle
with the triangularly shaped pipet.
9.- Put plate at 37C overnight inverted (agar up)
10.- Inoculate the minipreps
262
PCR METHOD TO CHECK BACTERIAL COLONIES OBTAINED FOLLOWING

TRANSFORMATION
(Suiza, C. Hetz, 2001)
REAGENTS
Reaction mix (final concentration in 25 l)
1 M primer sense
1M primer antisense
0.5U Taq polymerase (from Ciencias of U.Chile)
PCR Buffer
1x
2mM MgCl2
0.2 mM each one
of dNTPs
PROTOCOL
Pick a single colony from plate with cells transformed, using sterile pipette tip 20
l. Streak out on a fresh LB-agar plate and incubate O/N at 37C . Wash out the
same tip into a sterile Eppendorf tube (1.5 ml) with 20 l sterile nanopure water.
Transfer the tubes to a water bath that has been preheated to 95C for 2 min.
Rapidly transfer the tubes to an ice bath. Leave cells there for 5 minutes.
Spin at 13000 rpm for 5 min. Use the resulting supernatant for PCR reaction. If
your mix reaction is 25 l, use 5l of supernatant.
Licaralln Quevedo/ 2001
263
RNA ISOLATION USING TRI REAGENT
PROTOCOL
1. HOMOGENIZATION
Cells should be lysed directly in a culture dish. Pour off media, add TRI Reagent
(Molecular Research Center, Inc.) and pipette cell lysate up and down several times.
Use 1 ml of TRI Reagent per 10 cm2 of culture dish area. (5 - 10 x 106 animal cells).
Avoid washing cells before the addition of TRI Reagent as this may contribute to mRNA
degradation.
2. PHASE SEPARATION
Store the homogenate for 5 minutes at room temperature to permit complete

dissociation of nucleoprotein complexes.
Supplement the homogenate with 0.2 ml cold chloroform per 1 ml of TRI Reagent,
cover the samples tightly and shake vigorously for 15 seconds.
Store the resulting mixture at room temperature for 2-15 minutes and centrifuge at
13.000xg for 15 minutes at 4 oC.
Following centrifugation, the mixture separates into a lower red phenol-chloroform

phase, interphase and the colorless upper aqueous phase. RNA remains exclusively
in the aqueous phase whereas DNA and proteins are in the interphase and organic
phase.
3. RNA PRECIPITATION
Transfer the aqueous phase to a fresh tube.
Precipitate RNA from the aqueous phase by mixing with cold isopropanol. Use 0.5
ml of cold isopropanol per 1 ml of TRI Reagent used for the initial homogenization.
Store samples on ice for 5-10 minutes and centrifuge at 13.000xg for 8 minutes at 4
25oC.
RNA precipitate (often invisible before centrifugation) forms a gel-like or white

pellet on the side and bottom of the tube.
264
4. RNA WASH
Remove the supernatant and wash the RNA pellet (dissolve by pipetting up and
down) with a small volume of 75% cold ethanol first and then with the rest of total
volume and subsequently centrifuge at 13.000xg for 5 minutes at 4 - 25 oC.
Add at least 1 ml of 75% ethanol per 1 ml TRI Reagent used for the initial
homogenization.
5. RNA SOLUBILIZATION
Remove the ethanol wash and briefly air-dry the RNA pellet at 37C for 10 minutes.
It is important not to completely dry the RNA pellet as this will greatly decrease its
solubility. Do not dry RNA by centrifugation under vacuum.
Dissolve RNA with 20 l of DEPC water 0,1% and 0,3 l of RNase inhibitor by
passing the solution a few times through a pipette tip and incubating for 10-15
minutes at 55-60C. Water used for RNA solubilization should be made RNase-free
by diethyl pyrocarbonate (DEPC) treatment: under chemical hood add 1 ml of
diethylpyrocarbonate (DEPC) to 1 l of nanopure water, mix vigorously and leave
overnight at 37C. Autoclave the next day to destroy DEPC.
Established in Chile by Claudio Hetz, July
2001
265
HUMAN COX-2 SEMI-QUANTITATIVE RELATIVE RT-PCR (AMBION)

RT Reaction (20 l)
-Assemble RT-reaction on ice in a PCR tube:
Amount
2,5g
4 l
2 l
To 6 l
Component
Total RNA
dNTP mix (2,5 mM each)
Random primers
Nuclease free water
- Mix, spin briefly in centrifuge and heat 3 minutes at 70 C.

- Cool briefly (s) on ice, spin briefly, place again on ice.
- Add the remaining RT components:
Amount
4 l
1 l
0,5 l
Component
5X AMV-TR buffer
RNase inhibitor
AMV-TR
- Incubate at 42 C for one hour.

- Store the reaction at 20 C or proceed to amplification step.
PCR Reaction (50 l )
PCR cocktail (one reaction):
Amount
1 l
5 l
4 l
3 l
0,25 l
2 l
2 l
32,75 l
Component
RT Reaction
10X PCR Buffer
DNTPs (2,5 mM each)
MgCl2 (25 mM)
Taq Polymerase
Mix 18s PCR primer pair: 18s competimer
(3:7 ) 5 M each
COX-2 primers ( 5 M )
Nanopure water
- Mix, spin briefly.

- Place on ice.
- Add 35 l of mineral oil.
PCR Program:
94 C
5 min.
35 cycles of the following profile: - 94 C
266
30 sec.
- 59 C
- 72 C
72 C
4 C
30 sec.
30 sec.
10 min.
- Analyse results on 2% agarose gels by adding loading dye to 20 l of each reaction.

- The 18s primer set produces an amplicon of 495 bp.
- The COX-2 primers produces an amplicon of 297 bp.
Established in Chile by Daniela Galleguillos, July 2001
267
TRANSFORMATION OF CA-COMPETENT BACTERIA WITH LIGATED DNA
PROTOCOL
Thaw Ca-competent cells (DH5, XL1B or Bl21) on ice for 25 minutes.
Using sterile pipette tip, transfer 200 l of competent cells to a sterile Eppendorf
tube. Add DNA (no more than 50 ng in a volume of 10 l or less) . Mix gently by
flicking tube with finger nail. Store the tubes on ice for 30 minutes.
Transfer the tubes to a water bath that has been preheated to 42C and incubate for
exactly 30 seconds. Do not shake the tubes.
Rapidly transfer the tubes to an ice bath. Allow the cells to chill for 2 minutes.
Add 800l of SOC mediun without antibiotics. Incubate the cultures for 1 h in a
water bath set at 37C to allow the bacteria to recover and express the antibiotic
resistance marker encoded by the plasmid. Shake the cells gently in this period.
Transfer 200l of transformed competent cells onto agar LB with appropiate

antibiotic selection. Using a sterile bent glass rod, gently spread the transformatied
cells over the surface of the agar plate (100-mm plate). If you want more colonies
simply plate more of the culture medium with transformed bacteria. To easily
accommodate larger volumes, however, the plates must be pre-dried for about an
hour at 37oC in the incubator.
When ALL the liquid has been absorbedby the plate surface, invert the plates and
incubate at 37C. Colonies should appear in 12-20 hours, depending on the bacteria
and the selection marker.
Protocol from Maniatics books and

modificate by Licaralln Quevedo
(2001)
268
MAKING COMPETENT CELLS USING CACL2

(Modification of Inoue and cols. 1990)
REAGENTS
SOB medium (1l):

bacto-tryptone
bacto-yeast
NaCl
20g
5g
0.5 g
Adjust to final volume of 1 liter by adding ddH2O. Sterilize by autoclaving.
TB medium (1l):
10mM de Hepes o PiPES
15mM CaCl2
55mM MnCl2
250mM KCl
Add all reagents exept MnCl2; adjust to pH 6-7 with KOH, then add MnCl 2.
Sterilize by filtration (dont autoclave).
PROTOCOL
Grow bacteria (DH5, XL1B or Bl21) in small-scale culture (about 10 ml)

overnight at 25C, 245 rpm. Next day, add 5 ml of O/N culture to a final volume of
200 ml SOB-medium. Keep shaking until OD600 is 0.6.
Cool on ice for 15 minutes. Spin using 50 ml sterile tubes at 3000 rpm for 15 min.
Resuspend in 67 ml cooled TB solution. Keep on ice for 10 min. Spin using 50 ml

sterile tubes at 3000 rpm for 10 min.
Resuspend in 16 ml TB solution-cool. Add slowly 1.2 ml of DMSO (7%). Keep on

ice for 10 min. Pipette 400 l into each sterile eppendorf tubes. Flash freeze in a
dry-ice/methanol bath. Store at 70C.
Note: For centrifugation use the eppendorf centrifuge in Stutzins Laboratory
Licaralln Quevedo/ 2001
269
TRANSFECTION BY ELECTROPORATION OF NIH3T3 CELLS

Cells are grown in DMEM (high glucose) with 10% heat-inactivated fetal bovine
serum (FBS). 107 cells are diluted in 1ml DMEM (high glucose) with 20% heat-inactivated
FBS and placed in the sterile electroporation chamber (Disposable Electroporation
Chamber from Life Technologies (0.4cm inter-electrode gap) (Cat. 11601-028)). 80-100
g of DNA present in a volume of max. 150 l of H2O are added and cells are
electroporated with a pulse at 250V and capacitance 1180mF (Electroporator Cell-Porator
from Life Technologies (Cat. 71600-050)). The cells are left to rest after electroporation for
5 min. without any movement. Then cell viability is determined by the Trypan Blue
Exclusion method (should be around 60-70%, but not more than 80% = inefficient
transfection). The rest of the cells are transferred to two plates with DMEM containing20%
heat-inactivated serum and left at 37oC/5% CO2 o/n. The following day cells are changed
back again to regular DMEM (high glucose) medium supplemented with 10% FBS. This
protocol can be used for both transient and stable transfection experiments. In the latter
case, add appropriate antibiotic for selection.
Protocol established experimentally with the collaboration of Dr. Rosario Billeta (Programa
de Inmunologa, ICBM, U. de Chile)
270
ELECTROTRANSFECTION OF A20 CELLS

The goal of this protocol is try to obtain an efficient method to transfect the murine
B-lymphoma cell A20.
Materials:
Electroporator Cell-Porator from Life Technologies (Cat. 71600-050)
Disposable Electroporation Chamber (0.4cm inter-electrode gap) (Cat. 11601-028)
Rich A20 Media (DMEM-High Glucose, 20% FBS, 50 M -ME, PSN)
A20 Media (DMEM-High Glucose, 10% FBS, 50 M -ME, PSN)
A20 Cells
G418 (Clontech 8056-1)
Method:
1. Cells growing are counted to check for quantity and viability
2. Wash the cells twice with rich A20 media, at 1200 rpm for 7 minutes
3. Concentrate the cells to 10x106 /ml
4. Add 1 ml of cells to each disposable electroporator chamber and leave on ice for
5 minutes
5. Add the DNA, around 1 or 50 g and leave for another 5 minutes on ice. Try not
to add more than 100 l of DNA solution. The smallest volume possible should
always be employed.
6. Electroporate the cells on ice, with a capacitance of 1180 F and voltage of 281
Volts (use electroporator in Rosario Billetters lab)
7. Leave in the electroporator for 5 minutes without moving
8. Put on ice for another 10 minutes
9. Now take an aliquot and check viability again. The remaining cells are diluted in
10 ml of rich A20 media, and left at 37C over night
10. The following day, change cells back to normal A20 media. If you want to make
a stable transfection you must dilute the cells to obtain 1 or 100 cells/well and
put in the 96 well plate in 100 l of normal A20 media with 1.5 2 mg/ml of the
appropriate antibiotic.
11. The following week, check cells for viability and change the media.
.Established in Chile by P. Rojas/July 2001
271
ENDOFREE DNA MIDI PREP (R. BILLETAS VERSION)

1.- Pick a single colony from a freshly streaked selective plate. Inoculate a starter culture of
2-5 ml LB medium containing the appropriate antibiotic. Grow during 8 h at 37C with
shaking (300 rpm).(Use a tube or flask with a volume of at least 4 times the volume of the
culture)
2.- Dilute the starter culture 1/500 to 1/1000 into selective LB medium.Innoculate 200 ml.
Grow at 37C for 12-16 h. with vigorous shaking (300 rpm). (Use a tube or flask with a
volume of at least 4 times the volume of the culture).
3.- Centrifuge the culture in 50 ml conical tubes at 3500 rpm for 30 min. in rotor 224.
4.- Discard the supernatant. Dry the pellet as much as possible and vortex gently.
5.- Resuspend pellet in 4 ml solution I. Add lysozyme 2 mg/ml (160 l of 50 mg/ml
lysozyme stock solution) and incubate on ice for 30 min. Put all pellets from the 200 ml
culture together at this step.
6.- Add 8 ml solution II and incubate on ice for 10 min.
7.- Add 6 ml solution III and incubate on ice for 20 min.
8.- Centrifuge at 10000 rpm for 20 min. at 4C. Alternatively distribute the mix into as
many eppendorf tubes as necessary (approximately 12 eppendorf tubes with 1.5 ml each).
9.- Transfer the supernatant to a fresh tube or to fresh eppendorf tubes (alliquot into 750 l)
and add 750 l isopropanol at 20C. Incubate on ice for 10 min.
10.- Centrifuge at 10000 rpm for 10 min. at 4C.
11.- Collect the pellets/pellet and resuspend in a final volumen of 1 ml esterilee water. Once
resuspended, distribute the mililiter into 4 eppendof tubes (250 l each).
12.- Add 250 l of cold (4C) 4M LiCl. Incubate on ice for 5 min.
13.- Centrifuge at 10000 rpm for 5 min at 4C
14.- Collect the supernatant and alliquot into 8 eppendorf tubes (250 l each) and incubate
at 65C for 5 min.
15.- Dilute 1:1 with steril H2O (250 l:250 l)
16.- Add 1 vol. isopropanol to precipitate and incubate at 20C O/N
17.- Centrifuge at 10000 rpm for 10 min. at 4C
18.- Resuspend the pellet in 1 ml of esterilee nanopure H2O. Add 20 g/ml RNase (2 l of a
10 mg/ml stock). Incubate at room temperature for 30 min.
19.- Add 100 l of 10% Tritn X-100 at 4C to the tube with 1 ml.
20.- Incubate at 4C with vigorous shaking for 30 min.
21.- Incubate the tubes at 37C or until turbidity appears.
22.- Centrifuge for 2 min. at 13000 rpm.
272
23.- Transfer the superior aquous phase (aprox. 1 ml) to a fresh eppendorf tube
24.- Repeat steps 19-23 twice more. At this point you only get 800 l of the aqueous
phase.Divide into two tubes with 400 l each.
25.- Add 400 l of phenol:chloroform. Mix by inverting the tube.
26.- Centrifuge at 12000 rpm for 5 min.at 4C
27.- Transfer the supernatant ( 400 l approx.) to a fresh tube and add 1 ml of EtOH (-20C)
and 40 l of sodium acetate endofree. Incubate on ice for 10 min.
28.- Centrifuge at 12000 rpm for 10 min at 4C
29.- Wash the pellet with 200 l of 80% EtOH Endofree at 4C
30.- resuspend the pellet in endo free steril water
273
ENDOFREE DNA MAXI PREP WITH QIAGEN COLUMNS

This protocol is designed for purification of up to 500 g endotoxin free plasmid DNA.
Endotoxin free DNA will improve transfection into sensitive eukaryotic cells.
The maximum recommended culture volumes are 100 ml for low copy plasmids and 250 ml
for high-copy plasmids. Using these volumes, expected yields are 300-500 g for high copy
plasmids and 50-250 for low copy plasmids.
shaking (300 rpm).(Use a tube or flask with a volume of at least 4 times the volume of the
culture)
2.- Dilute the starter culture 1/500 to 1/1000 into selective LB medium. For high copy
plasmids inoculate 100 ml and for low copy plasmids inoculate 250 ml. Grow at 37C for
12-16 h. with vigorous shaking (300 rpm). (Use a tube or flask with a volume of at least 4
times the volume of the culture).
3.-Harvest the bacterial cells by centrifugation at 6000 x g for 15 min. at 4C (Pellets can be
frozen at this point if you wish to stop the protocol)
4.- Resuspend well the bacterial pellet in 10 ml Buffer P1 until no cell clumps remain. (Use
a vessel big enough to allow complete mixing of the lysis buffers. Make sure that RNase A
has been added to Buffer P1)
5.-Add 10 ml buffer P2, mix gently but thoroughly by inverting 4-6 times, and incubate at
room temperature for 5 min. (Do not vortex. Do not let the reaction proceed for more that 5
min.)
During the incubation prepare the qiafilter cartridge: screw the cap onto the nozzle of the
qiafilter maxi cartridge. Place the qiafilter cartridge into a convenient tube.
6.- Add 10 ml chilled buffer P3 to the lysate and mix immediately but gently by inverting 46 times. Proceed directly to step 7. (Do not incubate the lysate on ice. Transfer the lysate
into the qiafilter cartridge immediately in order to prevent later disruption of the precipitate
layer).
274
7.- Pour the lysate into the barrel of the qiafilter cartridge. Incubate at room temperature for
10 min. Do not insert the plunger!. (IMPORTANT: the 10 min. incubation is essential for
optimal performance of the qiafilter maxi cartridge. A precipitate should float to the top of
the solution. If, after 10 min, the precipitate has not floated to the top of the solution, use a
sterile pipet tip to dislodge it).
8.- Remove the cap from the qiafilter outlet nozzle. Gently insert the plunger into the
qiafilter maxi filter and cell lysate into a 50 ml tube. (Approximately 25 ml of the lysate is
generaly recovered after filtration).
9.- Add 2.5 ml buffer ER to the filtered lysate, mix by inverting the tube approximately 10
times, and incubate on ice for 30 min.
10.- Equilibrate a qiagen-tip 500 by applying 10 ml buffer QBT, and allow the column to
empty by gravity flow.
11.- Apply the filtered lysate from step 9 to the qiagen-tip and allow it to enter the resin by
gravity flow.
12.-Wash the qiagen-tip with 2 x 30 ml buffer QC. (For all subsequent steps use endotoxinfree plasticware)
13.-Elute DNA with 15 ml buffer QN
14.- Precipitate DNA by adding 10.5 ml (0.7 volumes) room temperature isopropanol to the
eluted DNA. Mix and centrifuge immediately at aprox. 15000 x g for 30 min. at 4C.
Carefully decant the supernatant.(15000 g correspond to 9500 rpm in a Beckman JS-13
rotor and 11000 rpm in a Sorvall SS34 rotor. Alternatively, disposable conical bottom
centrifuge tubes can be used for centrifugation at 5000 x g for 60 min at 4C. Be careful
when removing the supernatant because isopropanol pellets are more loosely attached to the
side of the tube).
15.-Wash DNA pellet with 5 ml of endotoxin-free, room temperature 70% ethanol
(prepared with the endo-free water supplied with the kit) and centrifuge at 15000 x g for 10
min. carefully decant the supernatant without disturbing the pellet. (The 70% ethanol
removes precipitated salt and replaces isopropanol with the more volatile ethanol, making
the DNA easier to redissolve).
275
16.- Air-dry the pellet for 5-10 min., and redissolve the DNA in a suitable volume of
endotoxin-free buffer TE. (Make sure to rinse the walls in order to recover all the DNA,
specially if glass tubes have been used. Do not resuspend the DNA by pipetting up and
down. It is easier to dissolve DNA under alkaline conditions rather then in acidic buffers).
17.- Determine the yield by measuring DNA concentration using an UV spectrophotometer.
Check the quality on an agarose gel.
276
PLASMID DNA MINIPREPS

1.- Pick a single colony from a freshly streaked selective plate. Inoculate a culture of 2-3 ml
LB medium containing the appropriate antibiotic for high copy plasmids. Alternatively,
grow up to 10 ml for low copy plasmids. ( LB culture medium is recommended because rich media,
such as terrific broth, produce very high cell densities that may overload the DNA purification system ).
Grow overnight (it has been observed that as a culture ages the DNA yield may begin to decrease due to
cell death and lysis within the culture. An A600 of 2.04.0 for high-copy-number plasmids ensures that
bacteria have reached the proper growth density for harvesting and plasmid DNA isolation )
at 37C with
shaking (300 rpm).(Use a tube or flask with a volume of at least 4 times the volume of the culture).
2.- Pellet the bacs for 5 min.by centrifugation at 10.000 x g. Discard supernatant and
remove liquid excess by inverting the tube on a paper towel for 2 minutes
3.- Resuspend the cells in 100 l solution I pipetting up and down. Add 1 mg/ml of
lysozyme (10 l of a 10 mg/ml stock solution). Incubate 5 minutes on ice. Make sure that the
pellet is completely dissolved and that there are no visible cell clumps. This is critical for a good plasmid
yield
4.- Add 200 l solution II. Mix carefully by inverting the tube several times (IT IS
IMPORTANT NOT TO VORTEX) or
until the mixture becomes clear. Incubate 5 minutes on ice.
5.- Add 150 l solution III. Mix gently by inverting the tube several times (IT IS IMPORTANT
NOT TO VORTEX).
At this step proteins and genomic DNA are precipitated. Incubar 5
minutes on ice.
6.- Centrifuge in a microfuge for 5 minutes at 13000 rpm.
7.- Transfer the supernatant to a new tube and add 100 g/ml RNasa A. Incubate 30
minutes at 37C.
8.- Add 100 l solution IV. Add 2 volumes of 20C ethanol. This step precipitates plasmid
DNA.
9.- Alternatively, you can add 2 volumes of phenol:chloroform (1:1). Mix well by inverting
the tube for 3 minutes and centrifuge 5 minutes at 13000 rpm.
10.- Collect the aqueous phase and transfer it to a new tube. Repeat the phenol:chloroform
extraction as in step 8.
11.-Collect the aqueous phase again and transfer it to a new tube. Add 200 l of
chloroform. Mix by inverting the tube for 3 minutes and centrifuge at 13000 rpm for 5
minutes.
12.- Transfer the aqueous phase to a new tube and add 2 vols of 20C ethanol. Incubate at
20C for 30 minutes and centrifuge at 13000 rpm for 20 minutes at 4C.
13.- Wash the pellet with by adding 300 l of 20C 70% ethanol. Centrifuge at 13000 rpm
for 10 minutes at 4C.
277
14.- Discard supernatant and dry the pellet.

15.- Resuspend in 100 l of water or TE. DNA is stable in water without addition of a buffer if stored
at 20C or below. DNA is stable at 4C in TE buffer. Be aware that for posterior reactions, EDTA present in
TE may interfere with enzyme activities
SOLUTIONS
Solution I: (10 ml)
25 mM Tris pH 7.5
250 l of 1 M Tris pH 7.5
10 mM EDTA pH 8.0
200 l of 0.5 M EDTA pH 8
lisozyme
10 l of 10 mg/ml stock
Solution II (10 ml)

0.2N NaOH:
660 l of 3M NaOH
1% SDS
1ml of 10% SDS
Solution III
3M Potassium acetate ph 4.8
Solution IV:
8 M Ammonium acetate (H2O)
TE:
10 mM Tris pH: 7.5
0.1 mM EDTA
278
DNA MINI PREPS WITH PROMEGA COLUMNS

Catalog no. A1330
1.- Pick a single colony from a freshly streaked selective plate. Inoculate a culture of 2-3 ml
LB medium containing the appropriate antibiotic for high copy plasmids. Alternatively,
grow up to 10 ml for low copy plasmids. (LB culture medium is recommended because rich media,
such as terrific broth, produce very high cell densities that may overload the DNA purification system ).
Grow overnight (it has been observed that as a culture ages the DNA yield may begin to decrease due to
cell death and lysis within the culture. An A600 of 2.04.0 for high-copy-number plasmids ensures that
bacteria have reached the proper growth density for harvesting and plasmid DNA isolation)
at 37C with
shaking (300 rpm).(Use a tube or flask with a volume of at least 4 times the volume of the culture).
2.- Pellet the bacs for 5 min.by centrifugation at 10.000 x g. Discard supernatant and
remove liquid excess by inverting the tube on a paper towel for 2 minutes.
3.-Resuspend the pellet with 250 l Cell Resuspension solution
4.- Add 250 l Cell Lysis solution to the resuspended cells. Invert 4 times to mix.
5.- Add 10 l Alkaline Phosphatase Solution; mix by inverting the tube 4 times. Incubate
for 5 min. at room temperature.
6.- Add 350 l Neutralization solution; mix by inverting the tube 4 times.
7.- Centrifuge at 13000 rpm in a microfuge for 10 minutes at room temperature. Collect the
supernatant and discard the pellet.
At this point you can perform the purification by centrifugation or by using a vacuum
manifold.
CENTRIFUGATION PROTOCOL
8.- Insert spin column into collection tube. Transfer the cleared lysate (850 l) from step 7
to the prepared spin column by decanting. Be careful not to disturb or transfer any of the
white precipitate with the supernatant
279
9.- Centrifuge at 13000 rpm for 1 min at room temperature. Discard flowthrough and
reinsert column into collection tube.
10.- Add 750 l of column wash solution previously diluted with 95% ethanol, to the spin
column.
11.- Centrifuge at 13000 rpm for 1 min at room temperature. Discard flowthrough and
reinsert column into collection tube.
12.- Repeat the wash procedure using 250 l of column wash solution
13.- Centrifuge at 13000 rpm for 2 min at room temperature.
14.- Transfer the spin column to a new sterile 1.5 ml microcentrifuge tube, being careful not
to transfer any of the column wash solution with the spin column. If it has column wash
solution associated with it, centrifuge again for 1 minute at maximum speed.
VACUUM PROTOCOL
8.- Attach one vacuum adapter with Luerlok fitting to one port of the manifold. Insert a
Transfer the cleared lysate (850 l) from step 7 to the prepared spin column by decanting.
Be careful not to disturb or transfer any of the white precipitate with the supernatant.
9.- Apply a vacuum to pull the liquid through the spin column. When all liquid has been
pulled through the column, release the vacuum.
10.- Add 750 l of column wash solution previously diluted with 95% ethanol, to the spin
column.
11.- Apply a vacuum to pull the column wash solution through the spin column. When all
the liquid has been pulled through the spin column, release the vacuum.
12.- Repeat the wash procedure, using 250 l of column wash solution. Apply a vacuum to
pull the liquid through the spin column.
13.- Dry the spin column by applying a vacuum for 10 minutes.
280
14.- Turn off the vacuum and transfer the spin column to a 2 ml collection tube. Centrifuge
at maximum speed for 2 minutes to remove any residual column wash solution. Discard the
2 ml collection tube and any liquid collected during this step.
COMMON STEPS FOR BOTH PROTOCOLS:
15.- Transfer the spin column to a new sterile 1.5 ml microcentrifuge tube.
16.- Elute the plasmid DNA by adding 100 l of nuclease free water to the spin column.
Centrifuge at maximum speed for 1 minute at room temperature in a microcentrifuge.
17.- After eluting the DNA, remove the assembly from the 1.5 ml microcentrifuge tube and
discard the spin column.
18.- DNA is stable in water without addition of a buffer if stored at 20C or below. DNA is
stable at 4C in TE buffer. To store the DNA in TE buffer, add 11 l of 10X TE buffer to the
100 l of eluted DNA. Be aware that for posterior reactions, EDTA present in TE may
interfere with enzyme activities.
NOTE: red coloured written products are supplied in the kit
281
DNA MIDI PREP WITH QIAGEN COLUMNS

Notes before starting: This protocol is designed for preparation of up to 100 g of high- or
low- copy plasmid or cosmid DNA. Low copy plasmids that have been amplified in the
presence of chloramphenicol should be treated as high-copy plasmids when choosing the
appropriate culture volumen. The maximum recommended cuture volumes are 25 ml for
high-copy plasmids and 100 ml for low-copy plasmids.
Add the provided RNase A solution to Buffer P1 before use.The final concentration is 100
g/ml.
Check Buffer P2 for SDS precipitation due to low storage temperatures. If necessary,
dissolve the SDS by warming to 37C. Pre-chill Buffer P3 to 4C.
shaking (300 rpm).(Use a tube or flask with a volume of at least 4 times the volume of the culture)
2.- Dilute the starter culture 1/500 to 1/1000 into selective LB medium. For high copy
plasmids inoculate 25 ml and for low copy plasmids inoculate 100 ml. Grow at 37C for
12-16 h. with vigorous shaking (300 rpm). (Use a tube or flask with a volume of at least 4 times the
volume of the culture).
3.-Harvest the bacterial cells by centrifugation at 6000 x g for 15 min. at 4C (Pellets can be
frozen at this point if you wish to stop the protocol)
4.- Resuspend well the bacterial pellet in 4 ml Buffer P1 until no cell clumps remain. (Use a
vessel big enough to allow complete mixing of the lysis buffers. Make sure that RNase A has been added to
Buffer P1. The bacteria should be resuspended completely by pipetting up and down until no cell clumps
remain)
5.-Add 4 ml Buffer P2, mix gently but thoroughly by inverting 4-6 times, and incubate at
room temperature for 5 min. (Do not vortex to avoid shearing of the genomic DNA. Do not let the
reaction proceed for more that 5 min.).
6.- Add 4 ml of chilled Buffer P3 to the lysate and mix immediately but gently by inverting
4-6 times and incubate on ice for 15 minutes. (Precipitation is enhanced by using chilled Buffer P3).
7.- Centrifuge at 20000 x g for 30 min. at 4C. Remove supernatant containing DNA
promptly (Centrifugation should be performed in non-glass tubes. 20000 x g corresponds to 12000 rpm in a
282
Beckman JA-17 rotor or 13000 rpm in a sorvall SS34 rotor). NOTE: Instead of centrifugation steps 7 and 8,
the lysate can be efficiently cleared by filtration using a QIAfilter Midi Cartridge.
8.- Re-centrifuge the supernatant at 20000 x g for 15 min. at 4C. Remove supernatant
containing plasmid DNA promptly (This second centrifugation step should be carried out to avoid
applying suspended or particulate material to the QIAGEN tip because this material can clog the QIAGEN-tip
reducing or eliminating gravity flow).
9.- Equilibrate a QIAGEN-tip 100 by applying 4 ml of Buffer QBT and allow the column
to empty by gravity flow (QIAGEN.tips can be left unattended since the flow of buffer will stop when
the meniscus reaches the upper frit in the column).
10.- Apply the supernatant from step 8 to the QIAGEN-tip and allow it to enter the resin by
gravity flow (if the supernatant is left too long before loading it onto the tip and becomes cloudy due to
further protein precipitation, it must be re-centrifuged or filtered before loading to prevent clogging of the
tip).
11.- Wash the QIAGEN-tip with 2 ml of Buffer QC (The first wash is sufficient to remove all
contaminants in the majority of plasmid DNA preparations. The second wash is particularly necessary when
large culture volumes or bacterial strains producing large amounts of carbohydrates are used.)
12.- Elute DNA with 5 ml of Buffer QF (Collect the eluate in 10 ml or 30 ml tubes. If you want to
stop the protocol at this point, store the eluate at 4C. Storage periods longer than overnight are not
recommended).
13.- Precipitate the DNA by adding 3.5 ml (0.7 volumes) of room temperature isopropanol
to the eluted DNA. Mix and centrifuge immediately at 15000 x g for 30 min. at 4C.
Carefully decant the supernatant (centrifugation is carried out at 4C to prevent overheating of the
sample. A centrifugal force of 15000 x g corresponds to 9500 rpm ina Beckman JS-13 rotor and 11000 rpm in
a Sorvall SS-34 rotor. Alternatively, conical bottom centrifuge tubes can be used for centrifugation at 5000 x g
for 60 min. at 4C. Isopropanol pellets are more loosely attached to the side of the tube than ethanol
precipitated pellets. Thus, care should be taken when removing the supernatant ).
14.- Wash DNA pellet with 2 ml of room temperature 70% ethanol and centrifuge at 15000
x g for 10 min. Carefully decant the supernatant without disturbing the pellet (Alternatively,
conical bottom centrifuge tubes can be used for centrifugation at 5000 x g for 60 min. at 4C.The 70% ethanol
removes precipitated salt and replaces isopropanol with the more volatile ethanol, making the DNA easier to
redissolve).
283
15.- Air-dry the pellet for 5-10 min. and redissolve the DNA in a suitable volume of water
or buffer (TE). (DNA is stable in water without addition of a buffer if stored at 20C or below. DNA is
stable at 4C in TE buffer. Be aware that for posterior reactions, EDTA present in TE may interfere with
enzyme activities).
284
DNA MIDI PREP WITH PROMEGA COLUMNS

Catalog no. A7640
Initial considerations: Start each midiprep with a 10-100 ml overnight culture of E. coli.
DNA yields may vary between 10 g and 220 g depending on the volumen of bacterial
culture, the plasmid copy number and the bacterial strain used. Up to 200 g of high copy
number plasmid DNA can be obtained from a 100 ml culture. When isolating DNA from
low copy number plasmids, it is reccomended to process 100 ml of bacterial culture.
1.- Pellet 10-100 ml of cells by centrifugation at 10000 x g for 10 min. at 4C. Pour off the
supernatant and blot the tube upside down on a paper towel to remove excess liquid.
2.- Completely resuspend the cell pellet in 3 ml of Cell resuspension solution. (To aid
resuspension, manually disrupt the pellet by pipetting until no clumps are visible. Complete resuspension is
critical for optimal yields).
3.- Add 3 ml of cell lysis solution and mix by inverting the tube four times. Do not vortex.
The cell suspension should clear immediately.
4.-Add 3ml neutralization solution and mix by inverting the tube 4 times (Alternatively, if
using an EndA+ strain, add 6 ml of neutralization solution, mix by inverting the tube 4 times, and incubate the
lysate at room temperature for 10 minutes).
5.- Centrifuge at 14000 x g for 15 min. at 4C. If a tight pellet has not formed by the end of
the centrifugation, centrifuge for another 15 min.
6.- Carefully decant the supernatant to a new centrifuge tube, avoiding the white
precipitate. Alternatively, transfer the cleared supernatant by filtering it through a miracloth,
filter paper or an autoclaved coffee filter, into the new centrifuge tube.
7.- Add 10 ml of resuspended DNA Purification Resin to the DNA solution. Mix by
swirling. (Extended incubation of the resin and lysate is not necessary. Do not allow the resin to remain in
contact with the lysate for longer than it takes to load the minicolumns).
8.- For each midiprep use one midicolumn. Insert the midicolumn tip into the vacuum
manifold port
285
9.- Transfer the resin/DNA mixture into the midicolumn. Apply a vacuum to pull the
resin/DNA mix into the midicolumn. When all of the sample has passed through the
column, break the vacuum at the source. (If using EndA+ strain, add 15 ml of 40% isopropanol/4.2 M
guanidine hydrochloride solution to each column).
10.-Add 15 ml of column wash solution to the midicolumn and apply a vacuum to draw the
solution through the midicolumn.
11.- Break the vacuum at the source and add another 15 ml of column wash solution to the
midicolumn. Reapply a vacuum to draw the solution through the midicolumn (The column
wash procedure may take up to 30 minutes).
12.- Dry the resin by continuing to draw a vacuum for 30 secs. After the solution has been
pulled through the column. DO NOT DRY THE RESIN FOR MORE THAN 30 SECONDS.
Remove the midicolumnfrom the vacuum source. Separate the reservoir from the
midicolumn by breaking or cutting with a sharp scissors as shown:
Cut column here
13.- Transfer the midicolumn to a 1.5 ml microcentrifuge tube and centrifuge the
midicolumn at 10000 x g in a microfuge for 2 minutes to remove any residual column wash
solution. Transfer the midicolumn to a new microcentrifuge tube.
14.- Add 300 l of preheated (65-70C) nuclease free water to the midicolumn and wait 1
minute. Elute the DNA by centrifuging the midicolumn at 10000 x g for 20 seconds in a
microfuge. Remove and discard the midicolumn.
15.- A white pellet of resin fines may be present in the final eluate. Whether visible or not,
it is important to separate the fines from the DNA. Centrifuge the sample at 10000 x g in a
microfuge for 5 minutes to pellet the fines. Carefully transfer the DNA-containing
supernatant to a clean microcentriguge tube.
286
16.- DNA is stable in water without addition of a buffer if stored at 20C or below. DNA is
stable at 4C in TE buffer. To store the DNA in TE buffer, add 30 l of 10X TE buffer to the
300 l of eluted DNA. Be aware that for posterior reactions, EDTA present in TE may
interfere with enzyme activities.
287
DNA MINI PREP WITH QIAGEN COLUMNS

Notes before starting: To ensure high yields of DNA, use no more than 3 ml LB culture for
high-copy plasmids. For low-copy plasmids use no more than 10 ml LB culture.
Chloramphenicol treated cultures of low-copy pasmids should be treated as high-copy
plasmids.
Add the provided RNase A solution to Buffer P1 before use.The final concentration is 100
g/ml.
Check Buffer P2 for SDS precipitation due to low storage temperatures. If necessary,
dissolve the SDS by warming to 37C. Pre-chill Buffer P3 to 4C.
1.- Pick a single colony from a freshly streaked selective plate. Inoculate up to 3 ml LB
medium containing the appropriate antibiotic for high copy number plasmids. 10 ml is the
maximum volumen to be inoculated for low-copy number plasmids. Grow overnight at
37C with shaking (300 rpm).(Use a tube or flask with a volume of at least 4 times the volume of the
culture).
2.- Harvest the bacterial cells by centrifugation at 13000 rpm for 5 min.
3.- Resuspend the bacterial pellet in 0.3 ml of Buffer P1(Bacteria should be resuspended
completely, leaving no cell clumps).
4.- Add 0.3 ml of Buffer P2, mix gently, and incubate at room temperature for 5 minutes
(mix by inversion and do not vortex as this will result in shearing of the chromosomal DNA. DO NOT
ALLOW THE REACTION TO PROCEED MORE THAN 5 MINUTES).
5.- Add 0.3 ml of chilled Buffer P3, mix immediately but gently, and incubate on ice for 5
minutes.
6.- Centrifuge at maximum speed in a microfuge for 10 minutes. Remove supernatant
promptly (if the supernatant is not clear, a second, shorter centrifugation shoud be carried out to avoid
applying any suspended or particulate material which could clog the column)
7.- Equilibrate a QIAGEN-tip 20 by applying 1 ml Buffer QBT and allow the column to
empty by gravity flow (QIAGEN.tips can be left unattended since the flow of buffer will stop when the
meniscus reaches the upper frit in the column).
288
8.- Apply the supernatant from step 6 to the QIAGEN-tip 20 and allow it to enter the resin
by gravity flow (if the supernatant is left too long before loading it onto the tip and becomes cloudy due to
further protein precipitation, it must be re-centrifuged or filtered before loading to prevent clogging of the
tip).
9.-Wash the QIAGEN-tip 20 with 4 x 1 ml Buffer QC (The first 2 ml are sufficient to remove all
contaminants in the majority of plasmid DNA preparations. The second 2 ml ensures complete removal of
contaminants and will also ensure consistent results in sequencing. The second 2ml is particularly necessary
when large culture volumes or bacterial strains producing large amounts of carbohydrates are used).
10.- Elute DNA with 0.8 ml Buffer QF.

11.- Precipitate DNA with 0.7 volumes of room temperature isopropanol. Centrifuge
immediately at 10000 rpm for 30 min. in a microfuge and carefully decant the supernatant.
12.- Wash DNA with 1 ml of 70% ethanol, air-dry for 5 minutes, and redissolve in a
suitable volume of water or buffer TE (The 70% ethanol removes precipitated salt and replaces
isopropanol with the more volatile ethanol, making the DNA easier to redissolve. DNA is stable in water
without addition of a buffer if stored at 20C or below. DNA is stable at 4C in TE buffer. Be aware that for
posterior reactions, EDTA present in TE may interfere with enzyme activities).
289
Protocols
Cell Communications Laboratory
Edited by Virginia Monardes. July, 2003.
290

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PROTOCOLS

CELL COMMUNICATIONS LABORATORY

Edited by Virginia Monardes. July, 2003

To prepare the sandwich:

4.) Place another 3M pad on top of the nitrocellulose NO bubbles! followed by

To remove the blot from the holder:

PURIFICATION OF IG FROM SERA USING SEPHAROSE-PROTEIN A

Dialysis tubing : Spectrapor 4, M.W. Cutoff 12-14.000

Glycine 0.1 M-HCl Buffer pH 3

30% acrylamide/bisacrylamide (38 : 2)

Swell the beads in 1x PBS and pull them in an appropriate column.

and immediately before use add 6.7 l of 30% H2O2.

9) Read in ELISA-reader at 450 nm.

used to dilute antigen coating solution

2x blocking solution 1xPBS, 5% milk powder, 0.05% Tween20

1xPBS, 2.5% milk powder, 0.05% Tween20 solution

Polyclonal anti mouse (anti rabbit etc.) IgG coupled to alkaline

1M Diethanolamin (pH=9.8), 1 mM MgCl2 (before

Plates: NUNC ImmunoPlates, MaxiSorp F96

1:50 in blocking solution.

- mouse IgM anti-caveolin-1 (monoclonal; Transduction Laboratories,

- rabbit anti-PKC (polyclonal; Sigma: P4334 (PKC), P8333 (PKC), P8458

1 hour at 37C protected from light.

Put 10 l of antibody solution on parafilm in a humidified box.

- anti rabbit- FITC (Jackson, 111-095-144)

COUPLING PEPTIDES TO CARRIER PROTEINS

MOUSE MONOCLONALE ANTIBODY AFTER PEPTIDE INJECTION

IMMUNOPRECIPITATION WITH T-CELL EXTRACTS

0.5% NP-40, 0.5% Deoxycholate, 50mM NaCl,

0.5% NP-40, 500mM NaCl, 10mM

Place 2.5ml of 10% Pansorbin into a round-bottomed Falcon

OVERLAY ASSAY WITH PKC

conc., stored at 4C, light-protected).

ANTIBODY PURIFICATION FROM HYBRIDOMA SUPERNATANTS

ANALYSIS OF APOPTOTIC CASPASE-3 (FLUOROMETRIC)......................................22

ANALYSIS OF APOPTOTIC CASPASE-3 (FLUOROMETRIC)

ANALYSIS OF APOPTOTIC DNA FRAGMENTATION ON AGAROSE GEL

M. Hunn/ Aug. 98 (acc. to P. Schneider, TP)

DNA CONTENT ASSAYS BY FLOW CYTOMETRY

Resuspend the cells in 200-250 l of PBS containing 2% FBS

9. Fluorescence emission of samples is analyzed by Flow Cell Cytometer (FACS;

control cells, 5% serum

IN SITU CASPASE-3/CASPASE-8 ASSAY BY FACS

Claudio Hetz, Jan 2002

VIABILITY ASSAYS BY FACS

Seed 100 l of A20 cells (0,5 x 106 cell/ml) in a 96 wells plate.

Note: For DNA content analysis:

Claudio Hetz, Jan 2002

ASSESSMENT OF CHROMATIN CONDENSATION AND MORPHOLOGICAL

Claudio Hetz, Jan 2002

QUANTIFICATION OF PHOSPHATIDYLSERINE EXPOSURE BY FACS

As controls and to calibrate the cytometer (compensation of fluorescence), stain the

DNA FRAGMENTATION ASSAY

CASPASE-3 ACTIVITY ASSAY (CHROMOGENIC)

C-CHOLESTEROL LABELING OF CELLS BEFORE FRACTIONATION ON

PREPARATION OF GST-PBD BEADS (FOR CDC42 AND RAC ASSAYS)..............73

0.1 M Tris-HCl pH 7.4

12, 1 and 0.06% TX-114 in TBS (Serva, Nr. 37243)