A LEARNERS GUIDE TO

SDS PAGE (Sodium dodecyl sulfate Poly

acrylamide gel electrophoresis)
By, PRATYUSH KUMAR DAS
04 July 2013

SDS PAGE (SODIUM DODECYL SULFATE POLY ACRYLAMIDE GEL ELECTROPHORESIS)

In their native form, proteins fold into a variety of shapes, some compact, some
elongated. The rate of migration of native proteins through a sieving medium is
therefore more a reflection of their relative compactness, and less an accurate measure
of molecular weight. Denaturing the proteins nullifies structural effects on mobility,
allowing separation on a true charge/mass ratio basis. It also separates subunits in
multimeric proteins, allowing analysis of large, complex aggregates.
The most commonly used denaturant is sodium dodecyl sulfate (SDS). SDS is an
amphipathic surfactant. It denatures proteins by binding to the protein chain with its
hydrocarbon tail, exposing normally buried regions and coating the protein chain with
surfactant molecules. The polar head group of SDS adds an additional benefit to the
use of this denaturant. Proteins solubilized in SDS bind the detergent uniformly along
their length to a level of 1.4 g SDS/g protein. This creates a charge/mass ratio which is
consistent between proteins. For this reason, separation on a polyacrylamide gel in the
presence of SDS occurs by mass alone.
SDS is the most commonly used detergent in protein electrophoresis. Treatment with
SDS creates a uniform charge to mass ratio between different proteins.
SDS PAGE offers a rapid and relatively accurate way to determine protein molecular
weights. Masses determined by SDS-PAGE are usually accurate within 5-10%,
although occasionally proteins may retain enough secondary structure or contain
sufficient charged groups to migrate anomalously. The migration of histones, which
carry a strong intrinsic charge, is an example of this phenomenon.

GEL PREPARATION (CONTINUOUS & DISCONTINUOUS BUFFER SYSTEMS)

Two categories of buffer systems are available for SDS PAGE: continuous and
discontinuous. Continuous systems use the same buffer in both the gel and tank. While
continuous gels are easy to prepare and give adequate resolution for some
applications, bands tend to be broader and resolution consequently poorer in these
gels. Discontinuous buffer systems employ different buffers for tank and gel, and often
two different buffers within the gel, with a third buffer in the tank. Discontinuous systems
concentrate, or stack the protein samples into a very narrow zone prior to separation,
which results in improved band sharpness and resolution.
In the classic SDS PAGE system developed by Laemmli, the gel is divided into an
upper stacking gel of low percentage (i.e. large pore size) and low pH (6.8) and a

resolving gel with a pH of 8.8 with much smaller pores. Both gels contain only Cl as the
mobile anion. The tank buffer has glycine as its anion, at a pH of 8.8. When
electrophoresis begins, glycine enters the stacking gel, where equilibrium favors the
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zwitterionic form with zero net charge. The glycine front moves slowly through the

stacking gel, lagging behind the strongly charged, smaller Cl ions. As these two current
carrying species separate, a region of low conductivity, with a consequent high voltage
drop, is created between them. This zone (a Kohlrausch discontinuity) sweeps the
proteins rapidly through the large pores of the stacking gel, collecting the sample and
depositing it at the top of the resolving gel in a focused narrow band. When the
Kohlrasch discontinuity enters the resolving gel, the increase in pH ionizes the glycine
so that it runs faster, dissipating the discontinuity. This allows the proteins to unstack
and separate through the small pore resolving gel.
PROCEDURE
Wiping/ Cleaning of Plates:
Glass plates are wiped with alcohol and white petroleum jelly/ melted agar is added to spacers
for attaching the two plates. check for leakage between the spacers.
Preparation of Buffers:
1. Stacking Gel Buffer
125 mM Tris-HCl; pH 6.8
0.1% SDS

To make a 4X Stock (500 ml):

30.35 g Tris base; pH'd to 6.8 with HCl.
20.0 ml 10% SDS (or 2.0 g solid)
~400 ml of ddH2O

2. Separating Gel Buffer

375 mM Tris-HCl; pH 8.8
0.1% SDS 20.0 ml

To make a 4X Stock (500ml)

91.0 g Tris base; pH'd to 8.8 with HCl
10% SDS (or 2.0 g solid)
~350 ml ddH2O

3.Electrophoresis Buffer
25 mM Tris; pH 8.3
250 mM Glycine
0.1% SDS
pH = 8.3 without adjustment

To make a 5X stock (1L):

15.1 g Tris base
72.0 g Glycine
5.0 g SDS
~850 ml ddH2O

4. Protein Gel Sample Loading Buffer

50 mM Tris-HCl; pH 6.8
2% SDS
10% Glycerol
1% -Mercaptoethanol
12.5 mM EDTA
0.02 % Bromophenol Blue

To make a 4X Stock (10 ml)

2.0 ml 1M Tris-HCl; pH 6.8
0.8 g SDS
4.0 ml 10% Glycerol
0.4 ml 14.7 M BME
1.0 ml 0.5 M EDTA
8 mg Bromophenol Blue

Preparation of Stock 30 % Acrylamide Solution:

The acrylamide stock is prepared by mixing dissolving acrylamide and bis-acrylamide in distilled
water in certain ratios so as to get the desired pore size. In general there are 3 different ratios
in which acrylamide & bis- acrylamide are mixed. The ratios are
1. Acylamide:Bis-Acrylamide = (19:1)
2. Acylamide:Bis-Acrylamide = (29:1)
3. Acylamide:Bis-Acrylamide = (37.5:1)

30% Acrylamide:Bis Solution (19:1)

Acrylamide
N,N'- methylenebisacrylamide
Add distilled H2O to make a final volume of

19 g
1g
66.6 ml

30% Acrylamide:Bis Solution (29:1)

Acrylamide
N,N'- methylenebisacrylamide
Add distilled H2O to make a final volume of

29 g
1g
100 ml

30% Acrylamide:Bis Solution (37.5:1)

Acrylamide
N,N'- methylenebisacrylamide
Add distilled H2O to make a final volume of

37.5 g
1g
128 ml

Heat the solution to 37oC to dissolve the chemicals.

Filter the solution through a 0.45 um-membrane filter.
Adjust the pH to less than 7.0.
Store the solution in dark bottles at room temperature for less than 3 months.

Preparation of Separating Gel:

2ml of distilled water, 2.5ml of separating gel buffer, 3.3ml of stock acrylamide solution are
added, then 10 microliter of TEMED,200 microliter of SDS and 100 microliter of APS are added
to it. Then the gel is immediately poured in between two plates and is left for some time for
solidification.
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Preparation of Stacking Gel:

2.5ml of distilled water, 1ml of stacking gel buffer, 2ml of stock acrylamide solution, and
10microlitre of TEMED, 100microlitre of SDS and 50 microliter of APS are added. Then it is
immediately poured in between the plates and the comb is placed for wells formation and it is
left for some time for solidification.
Preparation of Sample to be loaded into the wells:
30 micro litre of protein sample is added to 10 micro litre of loading buffer in eppendorf tube
and is boiled for 5 minutes in boiling water bath.
Running a Gel:
1.
2.
3.
4.
5.

The comb and the bottom spacer are removed carefully and is placed in SDS-PAGE tank.
The electrode buffer is poured in the tank.
Then the samples are loaded into the wells with the help of micropipette.
Power supply (100V) is switched on and is supplied for 2-3 hours.
The power supply is switched off, the plate is taken out from SDS-PAGE tank.

After Running a Gel:

1. The side spacers are removed. The two plates are separated with the help of spatula
and it is kept in to the staining buffer for overnight.
2. After incubation, the gel is transferred in to the destaining solution for 1-2 hours. Then
bands are observed.
Composition of Staining Solution/ Coomassie Stain (1 L):
0.1% Coomassie R250, 10% acetic acid, 40% methanol
Reagents needed:
1g

Coomassie R250

100 ml

glacial acetic acid

400 ml

methanol

500 ml

ddH2O

Directions:
1) Add 100 ml of glacial acetic acid to 500 ml of ddH2O.
2) Add 400 ml of methanol and mix.
3) Add 1g of Coomassie R250 dye and mix.
4) Filter to remove particulates (a coffee filter works great for this and is cheap)
5) Store at room temperature in a sealable container.

Composition of De-staining Solution (1L):

The composition is same as that of the staining solution just the addition of Coomassie R250 is
excluded.
OR
20% methanol, 10% acetic acid
Reagents needed:
200 ml

methanol

100 ml

glacial acetic acid

700 ml

ddH2O

Directions:
1) Add 100 ml of glacial acetic acid to 700 ml of ddH2O.
2) Add 200 ml of methanol and mix.
3) Store at room temperature in a sealable container.

How Does SDS PAGE Works???

The Role of SDS:
SDS is a detergent that is present in the SDS-PAGE sample buffer where, along with a bit of boiling,
and a reducing agent (normally DTT or B-ME to break down protein-protein disulphide bonds), it
disrupts the tertiary structure of proteins. This brings the folded proteins down to linear molecules.
SDS also coats the protein with a uniform negative charge, which masks the intrinsic charges on the
R-groups. SDS binds fairly uniformly to the linear proteins (around 1.4g SDS/ 1g protein), meaning
that the charge of the protein is now approximately proportional to its molecular weight.
SDS is also present in the gel to make sure that once the proteins are linearised and their charges
masked, they stay that way throughout the run.

The end results are

1) All proteins contain only primary structure

&

2) All proteins have a large negative charge which means they will all migrate towards the positive
pole when placed in an electric field.

The Role of Tris-HCl:

It is basically a biological buffer system that maintains the PH of both the gel system ( Stacking
& Separating at 6.8 and 8.8 respectively).
The Role of Electrode Buffer:
To conduct the current through from the anode to the cathode through the gel.
The Role of Glycine:
Glycine has a major role in separation of proteins. The stacking gel has a low concentration of
acrylamide and the running gel a higher concentration capable of retarding the movement of
the proteins.
Glycine can exist in three different charge states, positive, neutral or negative depending on the
pH.
When the power is turned on, the negatively-charged glycine ions in the pH 8.3 electrode
buffer are forced to enter the stacking gel, where the pH is 6.8. In this environment glycine
switches predominantly to the zwitterionic (neutrally charged) state. This loss of charge causes
them to move very slowly in the electric field.
The Cl- ions (from Tris-HCl) on the other hand, move much more quickly in the electric field and
they form an ion front that migrates ahead of the glycine. The separation of Cl- from the Tris
counter-ion (which is now moving towards the cathode) creates a narrow zone with a steep
voltage gradient that pulls the glycine along behind it, resulting in two narrowly separated
fronts of migrating ions; the highly mobile Cl- front, followed by the slower, mostly neutral
glycine front.
All of the proteins in the gel sample have an electrophoretic mobility that is intermediate
between the extreme of the mobility of the glycine and Cl- so when the two fronts sweep
through the sample well the proteins are concentrated into the narrow zone between the Cland glycine fronts.
This procession carries on until it hits the running gel, where the pH switches to 8.8. At this pH
the glycine molecules are mostly negatively charged and can migrate much faster than the
proteins. So the glycine front accelerates past the proteins, leaving them in the dust.

The result is that the proteins are dumped in a very narrow band at the interface of the stacking
and running gels and since the running gel has an increased acrylamide concentration, which
slows the movement of the proteins according to their size, the separation begins.
The Role of Glycerol:
Glycerol imparts density to the sample thereby enabling the sample to sink into the bottom of
the wells while loading. Instead of Glycerol, Sucrose can also be used.
The Role of -mercaptoethanol in sample buffer/loading buffer:
It breaks up the protein tertiary structure by breaking the disulphide bonds.
The Role of TEMED (N, N, N', N'-tetramethylethylenediamine) and APS (Ammonium
Persulphate):
(N2H8S2O8; mW: 228.2). APS is a source of free radicals and is often used as an initiator for gel
formation.
(C6H16N2; mW: 116.21). TEMED stabilizes free radicals and improves polymerization.
The Role of Acrylamide (C3H5NO; mW: 71.08):
When dissolved in water, spontaneous but slow auto polymerization takes place, joining
molecules together by head on tail fashion to form long single-chain polymers.
A solution of these polymer chains becomes viscous but does not form a gel, because the
chains simply slide over one another. Gel formation requires linking various chains together.
Acrylamide is a neurotoxin. It is also essential to store acrylamide in a cool dark and dry place to
reduce autopolymerisation and hydrolysis.
The size of the pores in the gel can be altered depending on the size of the proteins you want to
separate by changing the acrylamide concentration.

The Role of Bis-acrylamide (N,N'-Methylenebisacrylamide) (C7H10N2O2; mW: 154.17):

It is two acrylamide molecules joined head to head at their non-reactive ends. Considered as a
cross-linking agent, that can cross link two polyacrylamide chains to form a mesh like network
leading to formation of gel.

The Role of Bromophenol Blue:

Its known as the tracking dye and as clear from its name, it is used to track the sample while
the SDS PAGE is under process or running.
The Role of Coomassie Brilliant Blue R-250 (CBB)(C45H44N3NaO7S2; mW: 825.97):
Also known as staining dye, it is an anionic dye, which non-specifically binds to proteins. The
structure of CBB is predominantly non-polar, and it is usually used in methanolic solution
acidified with acetic acid. Proteins in the gel are fixed by acetic acid and simultaneously stained.

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