Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

Perform SDS-PAGE for separation of proteins according to a modified protocol (Laemmli, 1970; Matsudaira and Burgess, 1978). Sodium dodecyl sulfate, an anionic detergent, is used to completely suppress the native charge on the proteins. It gives them a large negative coat of detergent molecules. The SDS also interacts with the hydrophobic core of proteins, it causes a rapid and irreversible unfolding that linearizes the polypeptide chains which all now have a roughly equivalent charge/mass ratio. Therefore, in SDS-PAGE, the separation of proteins is on the basis of molecular weight (size) of the proteins.

Buffers and Reagents:

Lower Gel Stock Solution (1.0 M Tris-HCl, pH 8.8; 0.25% SDS)

60.6 g Tris-HCl

1.25 g SDS

Dissolve in H₂O, adjust the pH 8.8 with HCl and make the volume 500 ml with H₂O.

Upper Gel Stock Solution (0.5 M Tris-HCl, pH 6.8; 0.25% SDS)

15.2 g Tris-HCl

0.63 g SDS

Dissolve in H₂O, adjust the pH 6.8 with HCl and make the volume 250 ml with H₂O.

1X SDS Running Buffer (0.19 M Glycine; 25 mM Tris-HCl, pH 8.3; 0.1 % SDS)

7.2 g Glycine

1.5 g Tris-HCl

0.5 g SDS

Dissolve in H₂O, adjust the pH 8.3 and make the volume 500 ml with H₂O.

5X SDS Sample Buffer (0.25 M Tris-HCl, pH 6.8; 50% Glycerol; 5% SDS and 0.1% Bromophenol blue)

1.2 g Tris-HCl

20 ml Glycerol

10 ml of 20% SDS

0.04 g Bromophenol blue

Dissolve in H₂O, adjust the pH 6.8 and make the volume 40 ml with H₂O.

** CAUTION ** SDS powder is hazardous. Prepare solution in a ventilated fume hood. 

Protocol:

For casting gels, a system with vertically oriented glass plates with 1 mm spacer in between can be used. Prepare separating gel (small pore size gel) and stacking gel (large pore size gel) as first described by Ornstein, 1964.

Composition of separating and stacking gel:

Components	Separating gel	Separating gel	Stacking gel (4%) (ml)
	10% (ml)	17% (ml)
40% Acrylamide/ Bis acrylamide (37.5:1)	15	25.6	5.4
Tris HCl (1.0 M, pH 8.8)	22	22	-
Tris HCl (0.25 M, pH 6.8)	-	-	27
10% SDS	0.6	0.6	0.54
TEMED	0.12	0.12	0.108
10% APS	0.065	0.065	0.065
H2O	22	11.5	20.9

First pour the separating gel between glass plates and apply a layer of isopropanol on it. After polymerization of the separating gel, remove the layer of isopropanol and wash the polymerized gel with H₂O. Then pour the stacking gel on the top of the polymerized separating gel and insert the comb. When the gel solution gets solidified, place the gel in the electrophoresis chamber filled with the 1X SDS running buffer and remove the comb. Mix the protein sample to be electrophoresed with 5X SDS sample buffer and denature it completely by heating at 95^oC for 5 minutes. Then load the denatured protein samples along with the molecular weight marker proteins and perform the electrophoresis at 150 volts. Stop the current when the bromophenol blue tracking dye reaches at the end.

Molecular weight protein markers that can be used:

Protein name	Molecular weight (kDa)
β-Galactosidase	116.0
Bovine serum albumin	66.2
Lactate-dehydrogenase	45.0
Restriction endonuclease Bsp981	35.0
Lactoglobulin	18.0
Lysozyme	14.4