Polyacrylamide disc gel electrophoresis
Analysis of proteins in their native form is carried out in polyacrylamide buffer gel. The classical disc electrophoresis using cylindrical gels has been described by Davis in 1964. On such occasions as isoenzyme studies, protein purity checking etc., electrophoresis in buffer gels is an essential procedure. For instance, a purified protein may consist of four subunits. When this is analyzed in denaturing conditions on SDS gels this would show four separate bands even though the protein is pure. In such case as evidence of protein purity, the appearance of a single band on a gel could be shown by running a normal buffer gel.
The separation of native (non-denatured) proteins in this method is based on both the charge and size of the protein. Since the native proteins are large and exist in quaternary structure, a gel of low percentage of acrylamide is used.
This gel system consist of a 7.5% separating gel with a stacking gel. In disc electrophoresis, the gels are cast in long cylindrical glass tubes. Prior to pouring the gel solution, the bottom of the tube is sealed with dialysis membrane or parafilm.
Various solutions used are as follows: