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Certificate of Analysis
Anti-Rap1
(Affinity purified Rabbit polyclonal IgG)
Catalog # 07-916
Lot # 0604028058
Immunogen: KLH-conjugated, synthetic peptide
(KYDPTIEDSYRKQVEVDC) corresponding to amino
acids 31-48 human Rap1a.
Specificity: Recognizes recombinant human
Rap1a and Rap1b proteins, Mr 22kDa.
Species Cross-reactivity: Tested on Human,
Mouse, and Rat. Broad on sequence conservation,
broad species cross-reactivity is expected.
FOR RESEARCH USE ONLY
NOT FOR USE IN HUMANS
Quality Control Testing
Quality Control Testing
Affinity Precipitation Assay: This antibody
was tested using the Rap1 Activation Assay
Kit (Catalog # 17-321). 32.5µg (50µl of
slurry) of Ral GDS Rap Binding Domain
(RBD; Catalog # 14-455) precipitated GTP-
Rap1 from HEK293 lysates. The precipitated
GTP-Rap1 was detected by immunoblot
analysis using 1µg/ml of this lot of polyclonal
anti-Rap1.
General References:
1. Lin D., et al., Nat. Cell. Biol. 2: 540-547, 2000.
2. Izumi Y., et al., J. Cell. Biol. 143: 95-106, 1998.
email: techserv@upstate.com
www.upstate.com
Formulation: 100 µL of 0.5 mg/mL of affinity purified
rabbit polyclonal IgG in 0.1M PBS with 0.05% sodium
azide in 30% glycerol.
Storage and Stability: Stable for 2 years at -20°C
from date of shipment.
Handling Recommendations: Upon first thaw, and
prior to removing the cap, centrifuge the vial and
gently mix the solution. Aliquot into microcentrifuge
tubes and store at -20°C. Avoid repeated freeze/
thaw cycles, which may damage IgG and affect
product performance. Note: Variability in freezer
temperatures below -20°C may cause glycerol-
containing solutions to become frozen during storage
Additional Research Applications
Immunoblot Analysis: 1µg/ml of this antibody detected
Rap1 in Jurkat, 293 and 3T3 cell lysates.
Affinity Precipitation and Immunoblot Analysis:
Representative blot from a previous lot. Samples
were treated as described for affinity precipitation
assay. Lane 1: HEK293 lysate in Rap1
Activation Lysis Buffer, 2X (RLB: Catalog # 20-
220) preincubated with GDP prior to precipitation
with Ral GDS-RBD; Lane 2: HEK293 lysate in
RLB preincubated with GTPγS prior to
precipitation with Ral GDS-RBD. Proteins were
resolved by electrophoresis, transferred to
nitrocellulose and probed with anti-Rap1
(1µg/ml). Visualization was achieved using a
goat anti-rabbit secondary antibody conjugated to
HRP and a chemiluminescence detection
system. Arrow indicates Rap1 (~22kDa).
Page Two of Two
Catalog # 07-916
Lot # 0604028058

Immunoblot Protocol
Affinity Precipitation/Immunoblot Protocol

Note: It is strongly recommended that the entire procedure be read thoroughly before beginning the assay.

A. Stock Solutions

1. Rap1 Activation Lysis Buffer, 2X (Catalog # 20-
220): 50mM Tris-HCl, pH 7.4, 500mM NaCl,
1% NP40, 2.5mM MgCl2, and 10% glycerol.
Add 10µg/ml aprotinin and 10µg/ml leupeptin
right before application (RLB). Optionally, the
phosphatase inhibitors 25mM sodium fluoride
and 1mM sodium orthovanadate may also be
added to the buffer.
2. 100X GTPγS, 10mM (Catalog # 20-176): Use 5µl
of GTPγS for GTP-labeling of 0.5ml of cell lysate.
3. 100X GDP, 100mM (Catalog # 20-177): Use 5µl of
GDP for GDP-labeling of 0.5ml of cell lysate.
4. 1M MgCl2: Formulate using deionized water.
5. 0.5M EDTA, pH 8.0: Formulate using deionized
water.

B. Cell Culture and Extract Preparation

Note: G-Proteins hydrolyze bound GTP to GDP very rapidly. Performing all steps at 4°C or on ice may reduce
hydrolysis. It is best to use freshly prepared extracts, but extracts that have been snap frozen in liquid nitrogen
and stored at –70°C or lower may be used.

Adherent Cells
1. Culture cells to approximately 85-90% confluence, stimulating Rap1 activation as desired.
2. Remove culture media, rinse twice with ice-cold Tris-Buffered Saline (TBS).
3. Add ice-cold RLB (0.5-1ml per 150mm tissue culture plate) to rinsed cells in plates on ice.
4. Detach (and lyse) the cells from plates by scraping with a rubber policeman or cell scraper.
5. Transfer the lysates to microfuge tubes on ice. Proceed to Step 6.

Non-Adherent Cells
1. Culture cells and stimulate Rap1 activation as desired.
2. Pellet cells by gentle centrifugation (500xg), then rinse twice with ice-cold Tris-Buffered Saline (TBS).
3. Discard supernatant, and add ice-cold RLB to the cell pellet (0.5-1ml per 107 cells).
4. Lyse cells by repeated (4-5 times) pipetting.
5. Transfer the lysates to microfuge tubes on ice. Proceed to Step 6.

6. If nuclear lysis occurs, the extract may be very viscous (and difficult to pipette) due to released genomic
DNA. DNA may be sheared by passing the lysate through a 26-gauge syringe needle 3-4 times (prior
passage through larger-bore needles may be required first).
7. Clear lysates of insoluble cell debris by centrifugation (5 minutes, 14,000xg, 4(C).
8. Remove the extract (supernatant), and store aliquots on ice (for immediate use) or snap freeze in liquid
nitrogen. Frozen extracts may be stored at –70°C or lower (for long term).

C. GTPγS/GDP Loading for Positive and Negative Controls

Note: Samples that will not be loaded with GTPγS/GDP may be kept on ice during the loading of controls.

1. Aliquot 0.5ml of each cell extract to two microfuge tubes.

2. To each tube, add 10µl of 0.5M EDTA (to 10mM, final concentration).
3. Add 5µl of 100X GTPγS (to 100µM, final concentration) to one tube (positive control).
4. Add 5µl of 100X GDP (to 1mM, final concentration) to a second tube (negative control).
5. Incubate the tubes for 30 minutes at 30°C with agitation.
6. Stop loading by placing the tubes on ice and adding 32µl of 1M MgCl2 (to 60mM, final concentration).
Page Three of Four
Catalog # 07-916
Lot # 0604028058

D. Rap1 Pull-Down Assay

Note: The optimal conditions for a specific cell system may need to be determined empirically.

1. Aliquot 0.5ml of each cell extract to a microfuge tube.

2. Add 30-50µl (20-30µg) of the Ral GDS-RBD agarose slurry.
3. Incubate the reaction mixtures for 45 minutes at 4°C with gentle agitation.
4. Pellet the agarose beads by brief centrifugation (10 seconds, 14,000xg, 4°C).
5. Remove and discard the supernatant.
6. Wash the beads (add 0.5ml RLB, mix gently, pellet beads, remove RLB) 3 times with RLB. Take care to
minimize loss of beads when RLB is removed.
7. Resuspend the agarose beads in 40µl of 2X Laemmli reducing sample buffer and boil for 5 minutes. Pellet
the beads by brief centrifugation. Addition of 2µl of 1M dithiothreitol prior to boiling may improve release of
Rap1 from the beads.
8. Centrifuge briefly to collect condensate in the bottom of the tube.

E. Immunoblot Analysis and Detection

1. Mix the supernatant and the agarose pellet and load 25µl of the mixture per lane on an appropriate
polyacrylamide gel.
2. Perform SDS-PAGE and immunoblot to a nitrocellulose (or other) membrane.
3. Rinse the membrane twice with water.
4. Incubate the membrane 30 minutes at room temperature with agitation in freshly prepared PBS containing
3% nonfat dry milk (Catalog # 20-200), (PBS-MLK).
5. Incubate the membrane overnight at 4°C with agitation with anti-Rap1 diluted to 1µg/ml in freshly prepared
PBS-MLK.
6. Wash the membrane twice (5-10 minutes each) with PBS.
7. Incubate the membrane for 1 hour at room temperature with agitation in the appropriate secondary
antibody conjugate (a goat anti-rabbit-HRP conjugate is recommended, Catalog # 12-348, optimal dilution
must be empirically determined) in PBS-MLK.
8. Wash the membrane 3 times (5-10 minutes each) with PBS.
9. Use a detection method of choice. HRP conjugates are indicated for the secondary antibody, so an
appropriate luminol-based chemiluminescent HRP substrate solution is recommended (colorimetric
detection may be used, but sensitivity will be reduced, as compared with a chemiluminescent detection).
Page Four of Four
Catalog # 07-916
Lot # 0604028058

F. Abbreviated Procedure

Note: This abbreviated procedure is provided as a convenience to outline and facilitate performance of the
assay. It is recommended that a photocopy of the page be used. As a step is completed, it may be checked
off in the appropriate box. After completion of the assay, the page can then be incorporated into your lab
notebook.

Cell Culture and Extract Preparation

Adherent Cells
† 1. Culture cells, stimulate Rap1 activation, and then rinse twice with ice-cold TBS.
† 2. Add ice-cold RLB (0.5-1ml per 150mm tissue culture plate).
† 3. Detach and lyse cells by scraping, transfer lysates to microfuge tubes on ice, then proceed to Step 4.

Non-Adherent Cells
† 1. Culture cells, stimulate Rap1 activation, pellet cells, and then rinse twice with ice-cold TBS.
† 2. Discard supernatant, and add ice-cold RLB to the cell pellet (0.5-1ml per 107 cells).
† 3. Lyse cells by repeated pipetting, transfer lysates to microfuge tubes on ice, then proceed to Step 4.
† 4. If nuclear lysis occurs, shear DNA with a 26-gauge syringe needle.
† 5. Clarify lysates by centrifugation (5 minutes, 14,000xg, 4°C).
† 6. Remove the extract, and store aliquots on ice or snap freeze (and store at –70°C or lower).

GTPγS/GDP Loading for Positive and Negative Controls

† 1. Aliquot 0.5ml of each cell extract to two microfuge tubes.
† 2. Add 10µl of 0.5M EDTA.
† 3. Add 5µl of 100X GTPγ to the positive control tube.
† 4. Add 5µl of 100X GDP to the negative control tube.
† 5. Incubate for 30 minutes at 30°C.
† 6. Add 32µl of 1M MgCl2 to GTPγS/GDP controls.

Rap1 Pull-Down Assay

† 1. Aliquot 0.5ml of each cell extract to a microfuge tube.
† 2. Add 30-50µl of Ral GDS-RBD agarose to each tube.
† 3. Incubate 45 minutes at 4°C.
† 4. Pellet beads by centrifugation (10 seconds, 14,000xg, 4°C).
† 5. Remove and discard the supernatant.
† 6. Wash the beads 3 times with RLB.
† 7. Resuspend the beads in 40µl of 2X Laemmli buffer.
† 8. Add 2µl of 1M dithiothreitol, boil for 5 minutes, then pellet by centrifugation.

Immunoblot and Detection

† 1. Mix supernatant and beads, then load 25µl on a polyacrylamide gel, perform SDS-PAGE, and immuno-
blot to a membrane.
† 2. Rinse the membrane twice with water.
† 3. Incubate the membrane 30 minutes at room temperature in 3% PBS-MLK.
† 4. Incubate the membrane overnight at 4°C with anti-Rap1 (diluted to 1µg/ml in PBS-MLK).
† 5. Wash the membrane twice (5-10 minutes each) with PBS.
† 6. Incubate the membrane 1 hour at room temperature with secondary antibody conjugate in PBS-MLK.
† 7. Wash the membrane 3 times (5-10 minutes each) with PBS.
† 8. Use a detection method of choice.