SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS OF

PROTEINS
PRINCIPLE:

SDS PAGE is a widely used analytical technique for separation and characterization
of complex mixture of proteins and peptides and for the estimation of the relative
molecular weight. As in the principle of electrophoresis, it refers to the movement of
charged particles under the influence of an electric field. Proteins although
amphoteric polyvalent macromolecules, migrate in an electric field in a fashion
consistent with their net charge which is a conformation dependent characteristic,
protein electrophoresis is markedly affected by the pH, temperature, and ionic
composition of the electrophoretic medium. Almost all analytical electrophoreses of
proteins are carried out in polyacrylamide gels under conditions that ensure
dissociation of the proteins into their individual polypeptide subunits and that
minimize aggregation. Most commonly, the strongly anionic detergent SDS is used in
combination with a reducing agent and heat to dissociate the proteins before they
are loaded onto the gel. The denatured polypeptides bind SDS and become
negatively charged. Modifications to the polypeptide backbone, such as N- or O-
linked glycosylation, however, have a significant impact on the apparent molecular
weight. Thus, the apparent molecular weight of glycosylated proteins is not a true
reflection of the mass of the polypeptide chain. In most cases, SDS-polyacrylamide
gel electrophoresis is carried out with a discontinuous buffer system in which the
buffer in the reservoirs is of a pH and ionic strength different from that of the buffer
used to cast the gel. The ability of discontinuous buffer systems to concentrate all of
the complexes in the sample into a very small volume greatly increases the
resolution of SDS-polyacrylamide gels.

MECHANISM OF SEPARATION:

The discontinuous buffer system that is most widely used was originally devised by
Ornstein and Davis (1964). The sample and the stacking gel contain Tris-Cl (pH 6.8),
the upper and lower buffer reservoirs contain Tris-glycine (pH 8.3), and the resolving
gel contains Tris-Cl (pH 8.8). All components of the system contain 0.1% SDS as per
suggested by Laemmli (1970). The chloride ions in the sample and stacking gel form
the leading edge of the moving boundary, and the trailing edge is composed of
glycine molecules. Between the leading and trailing edges of the moving boundary is
a zone of lower conductivity and steeper voltage gradient, which sweeps the
polypeptides from the sample and deposits them on the surface of the resolving gel.
There the higher pH of the resolving gel favours the ionization of glycine, and the
resulting glycine ions migrate through the stacked polypeptides and travel through
the resolving gel immediately behind the chloride ions. Freed from the moving
boundary, the SDS-polypeptide complexes move through the resolving gel in a zone
of uniform voltage and pH and are separated according to size by sieving. The
sieving properties of the gel are determined by the size of the pores, which is a
function of the absolute concentrations of acrylamide and bisacrylamide used to cast
the gel.

CHEMICAL INGREDIENTS AND THEIR ROLES:

Chemical Ingredient Role

Polyacrylamide The sieving properties of the gel are determined by
the size of the pores, which is a function of the
absolute concentrations of acrylamide and
bisacrylamide used to cast the gel.
Tris  It has been used as a buffer because it is an
innocuous substance to most proteins. Its pKa is 8.3
(C4H11NO3; mW:121.14)  at 20°C, making it a very satisfactory buffer in the pH
range from roughly 7 to 9.

Glycine Glycine has been used as the source of trailing ion

or slow ion because its pKa is 9.69 and mobility of
(C2H5NO2; mW: 75.07)  glycinate are such that the effective mobility can be
set at a value below that of the slowest known
proteins of net negative charge in the pH range. The
minimum pH of this range is approximately 8.0.

Acrylamide In presence of free radicals generating system,

acrylamide monomers are activated into a free-
(C3H5NO; mW: 71.08)  radical state. These activated monomers polymerise
quickly and form long chain polymers.
Bisacrylamide

(N,N'-Methylene Bisacrylamide is the most frequently used cross

bisacrylamide) linking agent for poly acrylamide gels. Chemically it
is thought of having two-acrylamide molecules
(C7H10N2O2; mW: 154.17)  coupled head to head at their non-reactive ends.

Sodium Dodecyl Sulfate  SDS is the most common dissociating agent used to
denature native proteins to individual polypeptides.
(SDS) When a protein mixture is heated to 100°C in
presence of SDS, the detergent wraps around the
(C12H25NaO4S; mW: 88.38)  polypeptide backbone. It binds to polypeptides in a
constant weight ratio of 1.4 g/g of polypeptide. In this
process, the intrinsic charges of polypeptides
becomes negligible when compared to the negative
charges contributed by SDS. Thus polypeptides after
treatment becomes a rod like structure possessing a
uniform charge density, that is same net negative
charge per unit length. Mobilities of these proteins
will be a linear function of the logarithms of their
molecular weights.
Ammonium persulfate (APS) APS is an initiator for gel formation.
(N2H8S2O8; mW: 28.2)

Chemical polymerisation of acrylamide gel is used

TEMED  for SDS-PAGE. It can be initiated by ammonium
persulfate and the quaternary amine, N,N,N',N'-
(N, N, N', N'-tetramethyl tetramethylethylenediamine (TEMED).
ethylenediamine)
Increasing the amount of APS and TEMED results in
(C6H16N2; mW: 116.21) a decrease in the average polymer chain length, an
increase in gel turbidity and a decrease in gel
elasticity. Decreasing the amount of initiators shows
the reverse effect.

Bromophenol blue (BPB) BPB is the universal marker dye. BPB is the most
commonly employed tracking dye, because it is
(3',3",5',5" tetrabromo viable in alkali and neutral pH, it is a small molecule,
Phenolsulfonphthalein) 
it is ionisable and it is negatively charged above pH
(C19H10Br4O5S; mW: 69.99) 4.6 and hence moves towards the anode. Being a
small molecule it moves ahead of most proteins and
nucleic acids. As it reaches the anodic end of the
electrophoresis medium electrophoresis is stopped.
It can bind with proteins weakly and give blue colour.

Glycerol  It is a preservative and a weighing agent. Addition of

glycerol (20-30 or 50%) is often recommended for
(C3H8O3; mW: 92.09) the storage of enzymes. Glycerol maintains the
protein solution at very low temperature, without
freezing. It also helps to weigh down the sample into
the wells without being spread while loading.

Coomassie Brilliant Blue  CBB is the most popular protein stain. It is an anionic
R-250 (CBB) dye, which binds with proteins non-specifically. The
structure of CBB is predominantly non-polar.
(C45H44N3NaO7S2; Proteins in the gel are fixed by acetic acid and
mW: 825.97) simultaneously stained. The excess dye incorporated
in the gel can be removed by destaining with the
same solution but without the dye. The proteins are
detected as blue bands on a clear background.

Butanol  Water saturated butanol is used as an overlay

solution on the resolving gel. Some times isobutanol
(C4H10O; mW: 74.12) is also used.

Dithiothreitol Both are reducing agent used to disrupt disulfide

bonds to ensure the protein is fully denatured before
2-Mercaptoethanol  loading on the gel.
REAGENTS:

A. Acrylamide/bis (30% acrylamide mix)

Dissolve 29.0g acrylamide and 1.0g N'N'-bis-methylene-acrylamide in 100 ml

deionised water. Store it at 4°C in the dark bottle. (storing life: 30 days
maximum).Before use solution should be checked for proper traspirancy.(Non A/C)

B. 1.5 M Tris-HCl, pH 8.8

18.21g Tris base in 80 ml of deionised water. Adjust to pH 8.8 with HCl. Leave it
overnight and readjust the pH to 8.8 if required. Make up the final volume to 100ml
with deionised water. Store it at 4°C. Discard if any precipitation is seen. (A/C)

C. 1.0 M Tris-HCl, pH 6.8

12.14 g Tris base in 80 ml of deionised water. Adjust to pH 6.8 with HCl. Leave it
overnight and readjust the pH to 6.8 if required. Make up the final volume to 100ml
with deionised water. Store it at 4°C. Discard if any precipitation is seen. (A/C)

D. 10% SDS

Dissolve 10g SDS in 90 ml water with gentle stirring and bring to 100 ml with
deionised H2O. Prevent forming while mixing. (Non A/C)

E. 10% Ammonium Persulphate

Dissolve 0.01g of APS in 100µL deionised water. Use freshly prepared APS.

F. SDS gel-loading buffer (2X Stock)

125 mM Tris-Cl (pH 6.8)

200 mM dithiothreitol (DTT)

4% (w/v) SDS (electrophoresis grade)

0.1% bromophenol blue

20% (v/v) glycerol

Store 2x SDS gel-loading buffer lacking dithiothreitol at room temperature. Add

dithiothreitol from a 1M stock just before the buffer is used.

Note: Using 2X stock solution, prepare 1X SDS gel-loading buffer for use.

G. 5X electrode (Running) buffer, pH 8.3 (1X - Tris-glycine electrophoresis buffer)

25 mM Tris

250 mM glycine (electrophoresis grade) (pH 8.3)

0.1% (w/v) SDS

Prepare a 5x stock of electrophoresis buffer by dissolving 15.1 g of Tris base and

94g of glycine in 900ml of deionized H 2O. Then add 50 ml of a 10% (w/v) stock
solution of electrophoresis-grade SDS and adjust the volume to 1000 ml with H 2O.

H. Staining solution Coomassie Brilliant blue staining

225.0ml Methanol

225.0ml deionised water

50ml glacial acetic acid

1gm Coomassie Brilliant Blue (R-250)

Dissolve 1gm of CBB R-250 in methanol and keep on stirring over night. Then add
water and acetic acid to make the volume. Filter the solution through a Whatman No.
1 filter to remove any particulate matter.

I. Destaining Solution Coomassie Brilliant blue staining

225.0ml Methanol

225.0ml deionised water

50ml glacial acetic acid

Filter the solution through a Whatman No. 1 filter to remove any particulate matter.

Solutions for Preparing 12% resolving Gels for Tris-glycine SDS-Polyacrylamide Gel
Electrophoresis (Add in the order given)
Constituents Volume (For 5 ml gel)
Deionised H20 1.65ml
1.5M Tris (pH8.8) 1.25ml
10% SDS 50µl
30% acrylamide mix 2.0ml
10% ammonium persulfate 38 µl
TEMED (Stored in dark bottle) 7.5 µl

Solutions for Preparing 5% Stacking Gels for Tris-glycine SDS-polyacrylamide Gel

Electrophoresis (Add in the order given)
Constituents Volume (For 5 ml gel)
Deionised H20 3.4ml
1.0M Tris (pH6.8) 630µl
10% SDS 50µl
30% acrylamide mix 840µl
10% ammonium persulfate 38 µl
TEMED (Stored in dark bottle) 7.5 µl
Special Equipment:

Power supply (capable of supplying up to 500 V and 200 mA is needed)

Vertical electrophoresis apparatus (BIO RAD)

METHOD:

Pouring SDS-polyacrylamide Gels

1. Assemble the glass plates according to the manufacturer's instructions.

2. Determine the volume of the gel mold and prepare the required volume of
resolving gel.
3. Without delay, swirl the resolving gel mixture rapidly and proceed to the next
step.
4. Pour the acrylamide solution into the gap between the glass plates. Leave
sufficient space for the stacking gel (the length of the teeth of the comb plus 1
cm).
5. Overlay the gel with isobutanol or deionised water after 7-8 mins to prevent
intervention of oxygen in polymerization process. Place the gel in a vertical
position at room temperature.
6. After polymerization is complete (20 minutes), pour off the overlay and wash
the top of the gel several times with deionized H 2O to remove any
unpolymerized acrylamide. Drain as much fluid as possible from the top of the
gel, and then remove any remaining H2O with the edge of a paper towel.
7. Prepare the stacking gel as follows: In a disposable plastic tube, prepare the
appropriate volume of solution containing the desired concentration of
acrylamide, using the values given.
8. Without delay, swirl the mixture rapidly and proceed to the next step.
9. Pour the stacking gel solution directly onto the surface of the polymerized
resolving gel.
10. Immediately insert a clean Teflon comb into the stacking gel solution, being
careful to avoid trapping air bubbles. Add more stacking gel solution to fill the
spaces of the comb completely and proper sealing.
11. Place the gel in a vertical position at room temperature.

Preparation of Samples and Running the Gel

12. While the stacking gel is polymerizing, prepare the samples in the appropriate
volume of 1 X SDS gel-loading buffer (Mix well) and heat them to 90-100°C
for 3-5 minutes in dry bath or wet bath to denature the proteins.
13. Be sure to denature a sample containing marker proteins of known molecular
weights. Mixtures of appropriately sized polypeptides are available from
commercial sources. MBI fermantas marker are only denatured first time
before use after which they are just incubated at 37°C for 15 mins prior to
loading. (Better to check manufacturer instruction before use)
 14. After polymerization is complete (30 minutes), remove the Teflon comb
carefully. Use a squirt bottle to wash the wells immediately with deionized H 2O
to remove any unpolymerized acrylamide.
15. Mount the gel (with the plates) in the electrophoresis apparatus. Add Tris-
glycine electrophoresis buffer to the inner (full) and outer (submerge the gel
plate locking clips) reservoirs. Remove any bubbles that become trapped at
the bottom of the gel between the glass plates.
16. Load up to 10-14µl of each of the samples & 8µl of marker in a predetermined
order into the bottom of the wells.
17. Load an equal volume of 1X SDS gel-loading buffer into any wells that are
unused.
18. Attach the electrophoresis apparatus to an electric power supply. Apply a
constant voltage of 80V to the gel.
19. After the dye front has moved into the resolving gel, increase the constant
voltage to 100 V and run the gel until the bromophenol blue reaches the
bottom of the resolving gel. Then turn off the power supply. (time 1.5h-2.0h)
20. Remove the glass plates from the electrophoresis apparatus and place them
on a paper towel.
21. Use an extra gel spacer to carefully pry the plates apart. Mark the orientation
of the gel by cutting a corner from the bottom of the gel that is closest to the
leftmost well.
22. At this stage, the gel can be fixed, stained with Coomassie Brilliant Blue.

Staining SDS-polyacrylamide gels with Coomassie Brilliant Blue

23. Prepare the staining solution as above mentioned.

24. Immerse the gel in at least 5 volumes of staining solution and place on a
slowly rotating platform for a minimum of 2-3 hours at room temperature.
Remove the stain and save it for future use.
25. Destain the gel by soaking it in the destaining solution (methanol: acetic acid
solution) without the dye on a slowly rocking platform for 4-8 hours, changing
the destaining solution three or four times.
26. After destaining, store the gels in H2O in a sealed plastic bag.
27. Gels may be stored indefinitely without any diminution in the intensity of
staining; however, fixed polyacrylamide gels stored in H 2O will swell and may
distort during storage. To avoid this problem, store fixed gels in H 2O
containing 20% glycerol.
28. To make a permanent record, photograph the stained gel.

REFERENCES:
 Laemmli UK (August 1970). "Cleavage of structural proteins during the assembly of the head of
bacteriophage T4". Nature 227 (5259): 680–685. 

 http://en.wikipedia.org/w/index.php?title=SDS-PAGE&oldid=337482576

 Sambrook, Joseph. and Russell, David W. and Cold Spring Harbor Laboratory.... Edition, 3rd. ed.
A.8.40-A8.47.