Practical No : 10

Practical : Sodium Dodecyl Sulfate -Polyacrylamide Gel Electrophoresis [SDS-PAGE] of proteins

Objectives : To gain experience in polyacrylamide electrophoresis.

Introduction:

When charged molecules are placed in an electric field, they migrate toward either the positive (anode)
or negative (cathode) pole according to their charge. Electrophoresis is a technique that separates and
sometimes purifies macromolecules, especially proteins and nucleic acids, on the basis of their charge.
Proteins and nucleic acids are electrophoresed within a matrix or “gel”. Most commonly, the gel is cast
in the shape of a thin slab, with wells for loading the sample. The gel is immersed in an electrophoresis
buffer that contains ions to carry a current and some type of buffer to maintain the pH at a relatively
constant value.

Polyacrylamide gel electrophoresis (PAGE) is probably the most common analytical technique used to
separate and characterize proteins. Here, the matrix is polyacrylamide .A solution of acrylamide and
N,N’-methylene bisacrylamide is polymerized to form the matrix. Acrylamide alone forms linear
polymers.the bisacrylamide introduces cross links between polyacrylamide chains. The ‘pore size’ is
determined by the ratio of acrylamide to bisacrylamide, and by the concentration of acrylamide. High
ratios of bisacrylamide to acrylamide and high acrylamide concentrations result in matrices with smaller
pore size, and therefore, cause low electrophoretic mobility. Polymerizations of acrylamide and
bisacrylamide monomers are including by ammonium persulfate. This spontaneously decomposes to
form free radicals. Tetramethylethylenediamine [TEMED] a free radical stabilizer is generally included
to promote polymerization.

In SDS-polyacrylamide gel electrophoresis, sodium dodecyl sulfate (SDS) is included in the matrix,
buffer and samples. SDS is an anionic detergent. It has a negative head and a group and a lipophilic tail.
It binds non- covalently to proteins, with a stoichiometry of around one SDS molecule per two amino
acids. SDS causes proteins to denature and disassociate from each other (excluding covalent cross-
linking). It also confers negative charge. In the presence of SDS, the intrinsic charge of a protein is
masked. During SDS –PAGE, all proteins migrate toward the anode (the positively charged electrode).
SDS-treated proteins have very similar charge-to –mass ratios, and similar shapes. Therefore, the
technique can be used separate protein by size. It can also be used determine the relative molecular mass
of a protein.
Materials:

Mini gel system

1.5M Tris HCl. pH 8.80 [Resolving Gel buffer]

0.5M Tris HCl.pH 6.80[stacking gel buffer]

10%w/v SDS solution

Acrylamide Bisacrylamide 30.8% stock solution (30% acrylamide, 0.8% bisacylamide)

Fresh prepared 10% Ammoniumpersulfate solution

TEMED

Β-mercaptoethanol

Water saturated n-butanol

5x SDS PAGE sample butter

Electrode buffer

Procedure:

The gel casting unit was assembled according to the instructions supplied by the manufacturer.

Preparation of Resolving Gel

The resolving gel solution was prepared according to the table given below6.00ml of it was poured into
the chamber.

Stock Solution Resolving gel 7.5% Resolving gel 10%

1.5M Tris HCl. pH 8.80 2.50ml 2.50ml
10% w/v SDS stock 100µl 100μl
Acrylamide/ Bis Stock 30% 2.50ml 3.33ml
10% Ammoniumpersulfate 50μl 50µl
TEMED 5µl 5μl
Distilled water 4.80ml 4.02ml
Total volume 10ml 10ml
Small amount of water saturated n-butanol was added to form a thin layer over the resolving gel. The
resolving gel was allowed to polymerize for about 45min.

Preparation of the Stacking Gel

Stack Solution Stacking Gel

1.5M Tris HCl. pH 6.80 2.50ml
10% w/v SDS stock 100µl
Acrylamide/ Bis Stock 30% 1.33ml
10% Ammoniumpersulfate 50μl
TEMED 5µl
Distilled water 6.1ml
Total volume 10ml
The n-butanol layer was washed with some electrode buffer after complete polymerization. The comb
was placed and stacking gel was poured. Polymerization was allowed.

Fixing to the Running Tank

The cams were removed to release the running module and gels. Any residual acrylamide was washed.
The inner running module was placed into the running tank. The comb was carefully removed without
damaging the gel. The wells were immediately flushed with electrophoresis buffer using a syringe. The
appropriate volume of running buffer was added to the upper and lower chambers.

Sample loading

80μl of the sample was mixed with 20µl of the sample buffer and heated at 95°C for 3min to denature
proteins. About 40μl of this mixture was loaded using gel loading tip. 1,10,13,18,21,23,27 and 31
faction numbers from the ion exchange chromatography were used as the samples. The pipette tip was
1-2mm above the bottom of the well to minimize dilution of the sample and to keep the sample tight
layer during the sample loading process. The equivalent volume of sample buffer was used to fill unused
wells to maintain uniform electrical resistance across the gel. The safety lid was firmly replaced in order
make sure that the electrical connections form a good contact. The electrophoresis apparatus were
connected to the power pack and connected the power pack to the main supply. The controls were
adjusted to 150-200V, 50-85mA.

End of the run

The power supply setting was turned to zero. The main supply was turned off and disconnected the
power lid. The water supply was turned off when the unit had been cooled. The safely lid was removed.
The gel was unclamped and the plates were separated with a strong broad blade. The load was spread
over a wide area.

Washing the gel

The gel was washed briefly with distilled water, several times

Staining and distaining of the gel

The gels were immersed in the staining solution for at least 30min. the gel was removed from the
staining solution and immersed in the distaining solution until excess stain was washed off from the gel
and the bands were became visible.

Observations:

Figure 01: Schematic of electrophoretic protein separation in a polyacrylamide gel.

Conclusion

When proteins are separated in the presence of SDS and denaturing agents, they become fully denatured
and dissociate from each other. In addition, SDS binds non-covalently to proteins in a manner that
imparts:
  An overall negative charge on the proteins. Since SDS is negatively charged, it masks the
intrinsic charge of the protein it binds
 A similar charge-to-mass ratio for all proteins in a mixture, since SDS binds at a consistent rate
of 1.4 g SDS per 1g protein SDS (a stoichiometry of about one SDS molecule per two amino
acids)
 A long, rod-like conformation on the proteins instead of a complex tertiary shape (Figure 02)

As a result, the rate at which an SDS-coated protein migrates in a gel depends primarily on its size,
enabling molecular weight determination.

Figure 02: Effect of SDS on the conformation and charge of a protein.

Discussion

The separation of macromolecules in an electric field is called electrophoresis. A very common method
for separating proteins by electrophoresis uses a discontinuous polyacrylamide gel as a support medium
and sodium dodecyl sulfate (SDS) to denature the proteins. The method is called sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE).

SDS (also called lauryl sulfate) is an anionic detergent, meaning that when dissolved its molecules have
a net negative charge within a wide pH range. A polypeptide chain binds amounts of SDS in proportion
to its relative molecular mass. The negative charges on SDS destroy most of the complex structure of
proteins, and are strongly attracted toward an anode (positively-charged electrode) in an electric field.

Polyacrylamide gels restrain larger molecules from migrating as fast as smaller molecules. Because the
charge-to-mass ratio is nearly the same among SDS-denatured polypeptides, the final separation of
proteins is dependent almost entirely on the differences in relative molecular mass of polypeptides. In a
gel of uniform density the relative migration distance of a protein (Rf, the f as a subscript) is negatively
proportional to the log of its mass. If proteins of known mass are run simultaneously with the unknowns,
the relationship between Rf and mass can be plotted, and the masses of unknown proteins estimated.

Protein separation by SDS-PAGE can be used to estimate relative molecular mass, to determine the
relative abundance of major proteins in a sample, and to determine the distribution of proteins among
fractions. The purity of protein samples can be assessed and the progress of a fractionation or
purification procedure can be followed. Different staining methods can be used to detect rare proteins
and to learn something about their biochemical properties. Specialized techniques such as Western
blotting, two-dimensional electrophoresis, and peptide mapping can be used to detect extremely scarce
gene products, to find similarities among them, and to detect and separate isoenzymes of proteins.

Chemical ingredients

Mercaptoethanol

The β mercaptoethanol (or DTT) reduces any disulfide bridges present that are holding together the
protein tertiary structure. SDS (CH3-(CH2)10 - CH2OSO-Na+) is an anionic detergent and binds strongly
to, and denatures, the protein. Each protein in the mixture is therefore fully denatured by this treatment
and opens up into a rod-shaped structure with a series of negatively charged SDS molecules along the
polypeptide chain. On average, one SDS molecule binds for every two amino acid residues. The original
native charge on the molecule is therefore completely swamped by the SDS molecules. The β
mercaptoethanol is essential for disrupting disulfide bridges in proteins. However, exposure to oxygen in
the air means that the reducing power of β mercaptoethanol in the sample buffer decreases with time.

DTT is a reducing agent used to disrupt disulfide bonds to ensure the protein is fully denatured before
loading on the gel; ensuring the protein runs uniformly. Traditionally the toxic and less potent 2-
mercaptoethanol was used.

Figure 03: Chemical structure of β mercaptoethanol

Polyacrylamide Gel

It is a white crystalline powder. While dissolving in water, auto-polymerization of acrylamide takes

place. It is a slow spontaneous process by which acrylamide molecules join together by head on tail
fashion .Polyacylamide is a cross-linked polymer of Acrylamide. The length of the polymer chains is
dictated by the concentration of Acrylamide used, which is typically between 3.5 and 20%.
Polyacylamide gels are significantly more annoying to prepare than agarose gels. Because oxygen
inhibits the polymerization process, they must be poured between glass plates. Acrylamide is a potent
neurotoxin and should be handled with care. Wear disposable gloves when handling solutions of acryl
amide, and a mask when weighing out powder. Polyacrylamide is considered to be non-toxic, but
Polyacrylamide gels should also be handled with gloves due to the possible presence of free Acrylamide.
Polyacrylamide gels have a rather small range of separation, but very high resolving power.

Ammonium persulfate

Figure 04: Chemical structure of Ammonium persulfate

Ammonium persulfate is an initiator for gel formation. Ammonium persulfate 2S2O8 is a strong
oxidizing agent. It is very soluble in cold water, a large fall of temperature accompanying solution. It is
a radical initiator.

TEMED (N, N, N', N'-tetramethylethylenediamine)

Chemical polymerisation of acrylamide gel is used for SDS-PAGE. It can be initiated by ammonium
persulfate and the quaternary amine, N,N,N',N'-tetramethylethylenediamine (TEMED). The rate of
polymerisation and the properties of the resulting gel depends on the concentration of APS and TEMED.
Figure 05: Polyacrylamide gel formation

Sodium Dodecyl Sulfate

SDS is the most common dissociating agent used to denature native proteins to individual polypeptides.
When a protein mixture is heated to 100 °C in presence of SDS, the detergent wraps around the
polypeptide backbone. In this process, the intrinsic charges of polypeptides become negligible when
compared to the negative charges contributed by SDS. Thus polypeptides after treatment become 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.

Without SDS, different proteins with similar molecular weights would migrate differently due to
differences in mass charge ratio, as each protein has an isoelectric point and molecular weight particular
to its primary structure. This is known as Native PAGE. Adding SDS solves this problem, as it binds to
and unfolds the protein, giving a near uniform negative charge along the length of the polypeptide.

Figure 06: Chemical structure of sodium dodecyl sulfate

Tris (tris (hydroxy methyl) aminomethane)

It has been used as a buffer because it is an innocuous substance to most proteins. Its pKa is 8.3 at 20
°C, making it a very satisfactory buffer in the pH range from roughly 7 to 9.

Bisacrylamide (N,N'-Methylenebisacrylamide)

Bisacrylamide is the most frequently used cross linking agent for poly acrylamide gels. Chemically it is
thought of having two-acrylamide molecules coupled head to head at their non-reactive ends.

Figure 07: Chemical structure of Bisacrylamide

Chemicals for processing and visualization

The following chemicals are used for processing of the gel and the protein samples visualized in it.
Bromophenol blue

BPB is the universal marker dye. Proteins and nucleic acids are mostly colourless. When they are
subjected to electrophoresis, it is important to stop the run before they run off the gel. BPB is the most
commonly employed tracking dye, because it is viable in alkali and neutral pH, it is a small molecule, it
is ionisable and it is negatively charged above pH 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. Tracking Dye

The sample buffer also contains an ionizable tracking dye usually bromophenol blue that allows the
electrophoretic run to be monitored, and sucrose or glycerol which gives the sample solution density,
thus allowing the sample to settle easily through the electrophoresis buffer to the bottom when injected
into the loading well.

Glycerol

It is a preservative and a weighing agent. Addition of glycerol (20-30 or 50%) is often recommended for
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 dye, which binds with proteins non-specifically.
The structure of CBB is predominantly non-polar. So is usually used (0.025%) in methanolic solution
(40%) and acetic acid (7%). Proteins in the gel are fixed by acetic acid and 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. As SDS is also anionic, it may
interfere with staining process. Therefore, large volume of staining solution is recommended,
approximately ten times the volume of the gel.
Figure 08: Chemical structure of Coomassie Brilliant Blue

N-Butanol

Water saturated butanol is used as an overlay solution on the resolving gel.

References:

[1]Weber K, Osborn M (August 1969). The reliability of molecular weight determinations by dodecyl
sulfate-polyacrylamide gel electrophoresis.". J Biol Chem. 244 (16): 4406–4412. PMID 5806584.
[2]J. V. Maizel, Jr.; SDS polyacrylamide gel electrophoresis; 2000; TrendsBiochem. Sci.; 25; 590-592.
[3]Schaegger, H., and vonJagow, G. (1987). Tricine-Sodium dodecyl sulfate-Polyacrylamide Gel
Electrophoresis for the Separation of Proteins in the Range from 1 to 100 kDa. Anal.Biochem. 166, 368-
379
[4] Schägger H and von Jagow G (1987). Tricine-sodium dodecyl sulfate-polyacrylamide gel
electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166, 368 –
379.
[5] Wheeler D et al. (2004). Discontinuous buffer systems operative at pH 2.5 – 11.0, 0°C and 25°C.
Electrophoresis 25, 973–974.