Define the Disc Electrophoresis?
Disc electrophoresis has improved resolution and strong bands are obtained. It requires a two-gel system and several different buffers. The running gel in which the separation takes place is prepared and then overlaid by a short (1 cm), large- pored "Stacking" or "Spacer gel". The buffer in the lower reservoir and in the running gel is as described before, while that in the sample solution and in the stacking gel has a pH about two units less than that of the lower reservoir. When the current is switched on, the buffer ions from the upper reservoir migrate into the stacking gel as the stacking gel buffer ions migrate ahead of them. As this occurs, the upper reservoir buffer ions encounter a pH that is much lower than their pK.
They therefore assume their uncharged (or, in the case of glycine, zwitterionic) form and become electrophoretically immobile. This effect causes the macro-molecular ions to approach the running gel as stacks of very narrow (0.01 nm thick) bands or discs that are ordered according to their mobilities and lie between the migrating ions of the upper reservoir and those of the stacking gel. As the macromolecular ions enter the running gel, they slow down as a result of gel filtration effects. This permits the upper reservoir buffer ions to overtake the macromolecular bands and because of the running gel's higher pH, assume their fully charged form as they too enter the gel and electroporetic separation is achieved.
The compactness of the macromolecular bands entering the running gel greatly increases the resolution of the macromolecular separations. With a study of disc electrophoresis we come to an end of our study about electrophoresis. We hope you did not find the discussion presented in this section too technical. Just for your reference, we shall not be conducting any electrophoresis experiment during our laboratory work for this course, but it is important that you, as a student of dietetics, understand the concept and principle of electrophoresis clearly, since this is one important investigative technique used for research in the field of nutritional biochemistry. We suggest you now answer the questions included in the review question section included herewith. This will help you consolidate your understanding about electrophoresis.