Nonurea sodium dodecyl sulfate-polyacrylamide gel electrophoresis with high-molarity buffers for the separation of proteins and peptides

Abstract

A sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a discontinuous buffer system for separation of both peptides and proteins, which is a modification of the Laemmli system, is described. In the modified procedure, twofold higher concentrations of buffers in the separating gel and the running buffer solution allow superior resolution for peptides as small as M(r) 5000. The resolution of peptides was dependent on salt concentrations in the systems in which sodium chloride was partially substituted for Tris-HCl buffer and buffer concentrations were varied. In the stacking gel of the modified procedure, detection of peptides and SDS demonstrated a sharp stack of peptides at the trailing edge of the SDS stack. On the other hand, this SDS stack included peptides, forced them to diffuse, and produced a broad starting zone under Laemmli conditions. In addition, following expansion of the SDS stack impaired peptide resolution further in the separating gel. Accordingly, the different interaction with the SDS stack in the stacking process was found to produce different resolution of peptides in the electrophoretic procedures. The modified conditions have potential to provide a superior alternative to the Laemmli system for analysis of various proteins.