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Thursday, June 28, 2012

Disontinous SDS PAGE, Two Phase SDS-PAGE, SDS PAGE Principle


SDS stands for Sodium Dodecyl Sulphate and PAGE stands for polyacrylamide gel electrophoresis.
SDS PAGE is a technique used for separating proteins with difference in molecular weights.SDS PAGE can provide information about molecular weight, purity of particular protein preparation. The advantage of this technique is that it is simple and reproducible.

For SDS PAGE Protocol clcik here

SDS PAGE Principle

SDS PAGE works by sieving effect of the polyacrylamide gel, polyacrylamide gel can be prepared with wide range of pore size by varying arcylamide - bisacrylamide ratio.SDS an anionic detergent, which binds to the protein and imparts net negative charge to the proteins. 1.4grams of SDS binds per gram of protein.Mercaptoethanol assists the protein denaturation by reducing all disulfide bonds.Ammonium per sulfate acts as initiator for polymerization and TEMED (tetramethylethylenediamine) helps in polymerization.

When electric current is applied proteins move through the gel pores based on the molecular weight it gets separated.

For SDS PAGE Protocol clcik here

Why discontinuous gel system is used? / why gel with two different pH is used?

discontinous SDS PAGE

The two-phase, discontinuous gel system used in SDS-PAGE works because there are 3 major different charged species in an SDS gel: Cl- which has the highest mobility, then the SDS-protein complexes, and finally, the glycinate, which at pH 6.8, is the slowest. When the power gets switched on, the Cl-leads the way, followed by the protein and the glycinate essentially wedges the protein between itself and the Cl-. What this does is tightly stack the proteins into a nice, tight, thin starting zone--that's why the pH 6.8 gel is called a "stacking gel." When the complexes hit the higher pH running gel (pH 8.8), the glycinate becomes more fully ionized and its mobility increases. It now overtakes the protein-SDS complexes, follows the Cl-, and the two species leave the proteins behind. The proteins keep moving towards the anode, all with the same electrical mobility, since due to the SDS, they all have identical charge per unit mass. However, without a following charged species to keep them stacked together, they are now free to start separating based on their relative sizes.

For SDS PAGE Protocol clcik here