Question

Briefly explain the principle of SDS-polyacrylamide gel electrophoresis. What is the purpose of the SDS? Which proteins midrate slowest by SDS-PAGE?

Answer 1

Principle of SDS-polyacrylamide gel electrophoresis:

If the proteins are denatured and put into an electric field, they will all move towards the positive pole at the same rate, with no separation by size. So we need to put the proteins into an environment that will allow different sized proteins to move at different rates. The environment of choice is polyacrylamide, which is a polymer of acrylamide monomers. When this polymer is formed, it turns into a gel and we will use electricity to pull the proteins through the gel so the entire process is called polyacrylamide gel electrophoresis (PAGE).For proteins, sodium dodecyl sulfate (SDS) is an anionic detergent applied to protein samples to linearize proteins and to impart a negative charge to linearized proteins. This procedure is called SDS-PAGE.

Purpose of the SDS:

In most proteins, the binding of SDS to the polypeptide chain imparts an even distribution of charge per unit mass, thereby resulting in a fractionation by approximate size during electrophoresis.

Proteins that midrate slowest by SDS-PAGE:

Proteins that have a greater hydrophobic content, for instance many membrane proteins, and those that interact with surfactants in their native environment, are intrinsically harder to treat accurately using this method, due to the greater variability in the ratio of bound SDS.