Experimental Validation of the Predicted Properties of a Multiphasic Buffer System Applied to Polyacrylamide Gel Electrophoresis

Abstract

The properties of a buffer system predicted by T. M. Jovin's theory of multiphasic (discontinuous) buffer systems have been tested experimentally both in free solution and in polyacrylamide gels. The most widely applied multiphasic buffer system, viz., the Tris-glycine system of Ornstein and Davis was used for this purpose. The properties of this system were computed on the basis of the Jovin theory. The pH, specific conductance, and boundary displacement were measured in the buffers corresponding to the original and the operative buffers of both the stacking (upper) gel and the separation (lower) gel in polyacrylamide gel electrophoresis. Values of pH and specific conductance in the absence of gels were found to be in reasonable agreement with theory. In polyacrylamide gels, after removal of residual reactants of the polymerization reaction by equilibration with the appropriate buffer, there was good agreement between observed and predicted values of pH and boundary displacement. However, the observed values for specific conductance were lower than predicted. The effects of pre-electrophoresis and of gel concentration on pH, conductance, and boundary displacement were also studied. A new method has been developed for formation of stable pH gradients in polyacrylamide gel electrophoresis using multiphasic buffer systems. A concentration and pH gradient in the upper (stacking) gel as formed results in a pH gradient in the operative stacking gel (after passage of the stack). This pH gradient is stable for at least 8 h in the system investigated, and may be applicable to protein fractionation.