SDS-PAGE

Sodium dodecyl sulphate polyacrylaminde gel electrophoresis (SDS–PAGE) was performed to separate and observe the protein pattern of the sample by the method of Laemlli (1970).

Principle

SDS-PAGE is the most widely used method to resolve protein according to their molecular weight using, acrylamide and the cross linker agent N,N- methylene-bis-acrylamide, in the presence of the free radical and a catalyst (ammonium per sulphate and TEMED). They get polymerized into a uniform three-dimensional nework with pores which allow the electrophoretic mobility of protein to be resolved. The porosity of gel is determined by the relative proportion of acrylamide to bis acrylamide. In the method, the protein, irrespective of their initial changes are conferred uniform negative charges by sodium dodecyl sulphate. SDS is an anionic detergent which strongly binds to denature proteins. The multi subunit proteins are separated into their individual polypeptide components and the polypeptides are rendered linear by the concentrated effect of heat, SDS and β-mercaptoethanol. The protein which acquires negative charges and linear nature as mentioned above are electrophoresed under basic pH toward anode. After electrophoresis the gel may either be stained with Coomassie Brilliant blue (a stain which has a affinity to proteins) or silver staining.

Reagents

Preparation of stock solution and buffers

1. 30% Acrylamide

a. Acrylamide : 29.2 g

b. N,N’- methylene-bis-acrylamide : 0.8 g

Add water, dissolve and make upto 100ml and filter with Whatmann No.1 filter paper.

2. Separating gel buffer

a. Tris–HCl : 1.5 M, pH 8.8

Take 18.171 g of Tris. Dissolve in 60 ml of water and adjust the pH to 8.8 with HCl and make upto 100ml with water.

3. Stacking gel buffer

a. Tris–HCl : 1 M, pH 6.8

Take 6.057 g of Tris. Dissolve in 60 ml of water and adjust the pH to 6.8 with HCl and make upto 100 ml with water.

4. 10% SDS solution: 1 g of SDS on 10 ml of distilled water.

N,N,N’N’ - Tetra methylethlene diamine (TEMED).

Ammonium per sulphate : 1 g of APS in 10 ml of distilled water.

Electrophoresis buffer

a. Tris : 25 mM, pH 8.3

b. Glycine : 250 mM, pH 8.3

c. SDS : 0.1%

Dissolve in minimum amount of water (500 ml) and then add SDS. Allow to settle and dissolve. Then make it up to 2.5 litres.

Sample buffer 4X: 5.0 ml

Tris (1M, pH 6.8) : 2.1 ml

20% SDS : 100 mg

Glycerol (100%) : 1.0 ml

β-mercaptoethanol : 0.5 ml

Bromophenol blue : 2.5 mg

Distilled water : 0.4 ml

Staining solution (100 ml)

· Alcohol : 40%

· Acetic acid : 10%

· Coomassie brilliant blue : 250 mg

· Distilled water : 50%

Destaining solution (100ml)

· Alcohol : 50%

· Acetic acid : 10%

· Distilled water : 40%

Procedure

Preparation of gel

· The glass plates were washed in warm detergent solution, rinsed subsequently in tap water, deoinized water and ethanol and dried.

· The unnotched outer plate was laid on the table and Vaseline (or grease) was coated.

· Spacer strips was arranged appropriately at the sides and bottom of the plate.

· The notched inner plate was laid in position, resting on the spacer strips and arrangement was mounted vertically.

· Sealing was done properly without any leakage.

The volume of the gel solution required for making separating gel was calculated as follows :

Reagents	Volume (ml) required for gel
	8%	10%	12%
Water	9.3	7.9	6.6
30% Acrylamide mix	5.3	6.7	8.0
1.5 M Tris (pH 8.8)	5.0	5.0	5.0
10% SDS	0.2	0.2	0.2
10% APS	0.2	0.2	0.2
TEMED	0.012	0.008	0.008

· Ammonium persulphate and TEMED was added just prior to the pouring of the gel. The solution was mixed well and poured into the space between two plates leaving an inch of the upper space unfilled.

· Water was carefully laid over the surface of the poured gel mixture to avoid air oxidation.

· The gel mixture was allowed to polymerize, undisturbed at room temperature for 60 min.

· In the mean time gel mixture for stacking gel was prepared (the reagent in the following table yield 10 ml of 5% solution after the addition of APS and TEMED).

Reagent	Volume (ml) required for gel
Water	6.8
30% Acrylamide mix	1.7
1 M Tris–HCl pH 6.8	1.25
10% SDS	0.1
10% APS	0.1
TEMED	0.01

· After the separating gel was polymerized the over-laid water was removed carefully with a filter paper.

· The stacking gel was prepared and poured on top of the separating gel, immediately insert the ‘comb’ between the plates and allowed it to polymerize.

Preparation of protein sample

· The required volume of sample buffer was added to protein sample and they were loaded (the final concentration of the sample buffer in the prepared sample should come to 1X buffer).

· The samples were incubated for 5 min in a boiling water bath prior to loading.

· When polymerization was completed the comb was removed and the lower spacer strip was carefully removed. The vaseline (or grease) from the bottom was removed with a piece of tissue paper.

· The gel was attached to the electrophoresis tank using appropriate clips/clamps.

· The lower reservoir was filled with 1X electrophoresis buffer, using a bent Pasteur or syringe needle to remove any air bubble trapped beneath the bottom of the gel.

· The protein samples were loaded using a micropipette and the wells were completely and carefully filled with 1X electrophoresis buffer.

· The electrodes were connected to a power pack.

· The gel was run at constant current and 20 mA 100 V for 4–6 h at room temperature.

· Electrophoretic mobility of the sample was determined by the bromophenol blue front.

· At the end of the run the power pack was switched off.

· The gel and the plates were laid flat on the table and a corner of the upper glass plate was lifted on the table and a corner of the upper glass plate was lifted up and the gel was carefully removed.

Staining of the gel

· The gel was fixed in 10% TCA for 5 min.

· The gel was submerged in the staining solution for 2–4 h.

· After staining the gel was submerged in the destaining solution in order to remove the background staining.

· When the background stain was removed the protein bands appeared clear, then the gel was stored in 7% acetic acid and photographed.

Reference:

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.