Question

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

1. I would like to know the functions of resolving gel and stacking gel in SDS - PAGE analysis.

2. 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. )

We would appreciate your detailed explanation of each question. 

(Please Typing)

Answer 1

Let us first understand what this process is.

SDS-PAGE (sodium dodecyl sulphate- polyacrylamide gel electrophoresis) is a method to separate proteins by their apparent molecular weight. The proteins are denatured in a solution containing SDS and agents to break disulphides bonds. This means that the protein forms an unfolded linear molecule that becomes coated in the negatively charged SDS. The protein solution is then applied to the gel and a voltage applied, which causes the negatively charged proteins to migrate towards the anode. The gel acts as a sieve in that large proteins get trapped and move slowly through the pores of the gel matrix, while the small proteins move quickly. The net result is the proteins get separated based on their apparent molecular weights with the largest (heaviest) at the top of the gel (where the solution was applied) and the smallest (lightest) at the bottom.

Answer for point 1)

Stacking gel basically concentrates all the protein in one band and stacking gel has same pore size, so what it does is to bring all the protein in one line before entering the resolving gel. Imagine it as a race. Before the start of race all the participants needs to be placed exactly besides eachother , not before or after one another. And the resolving gel resolves all the protein based on its mol. wt.

Answer for point 2)

SDS-PAGE gel uses 3 buffers

Tris- Gly (8.3), Tris-Cl (pH 6.8) & Tris-Cl (8.8)

The Tris-Cl buffers are present in the stacking & resolving gels respectively. The Tris-Gly is the buffer used for running the apparatus. In Tris-Gly @ pH 8.3 the glycine exists as a –ve charge and moves towards the positive electrode. So this –vely charged Gly enters the stacking gel. In the stacking gel, the pH changes to 6.8 where Gly exists in zwitter-ionic form. Now Gly moves slowly but the Cl- (from Tris-Cl) moves fast and reaches the interface of resolving and stacking gel first. So in the stacking gel the Cl- forms the leading front & glycine forms the back-front with proteins in between these fronts. Now apart from the electric field provided by the electrodes, another electric field develops due to differential movement of Cl- and Gly ions. The two electric fields are in opposite directions and being different in magnitude, help the –vely charged proteins to stack at the interface of two gels. While entering resolving gel, as pH changes again, the Gly becomes –vely charge again and moves into the resolving gel quickly, effectively pushing the proteins into the resolving gel also. In the absence of the stacking gel, this push will not be there. So all proteins will move into the gel at their own pace & give rise to a smear. But if stacking gel is present, all proteins in the sample get stacked & are pushed into the gel at the same time, to start resolving. Now in the resolving gel, the proteins separate based on molecular mass.