Question

This is a question about the SDS - PAGE method, one of the protein analysis methods.

I would like to know the various reagents used in manufacturing resolving gel and stacking gel in SDS - PAGE analysis and their role. ( Please tell us in detail the role of each reagent. )

30% Acrylamide/bis

0.5M Tris-HCl, pH 6.8

1.5M Tris-HCl, pH 8.8

10% SDS

diH2O

TEMED

10% APS

We would appreciate your detailed explanation.

Please typing.

Answer 1

30 % acrylamide

in SDS- PAGE bis acrylamide is used to create crosslinks between acrylamide to form polyacrylamide gel. the porous character is depends on the ratio of bis acrylamide to acrylamide.

0.5M tris HCL

this is used to prepare stacking gel in sds page. 6g of tris bade is dissolved in 80ml dist. water and ph is adjusted to 6.8 using 6N Hcl . it is made upto 100 ml.

1.5M HCL

this is used to prepare resolving gel in sds page. dissolve 18.15 g of tris base into 80 ml of distilled water and adjust ph to 8.8 by adding 6N HCL. final is made upto 100 ml.

10% SDS

SDS is acting as surfactant. they are usually the denature the proteins. it disrupt the tertiary structure of proteins. the common reducing agent DTT breakdown protein - protein disulphide bonds

TEMED

it is a free radical stabilizer . it is generally promoting polymerization.  

APS

it is a oxidizing agent. it is used to catalyze the polymerization of acrylamide and bis acrylamide