Two-stage methods for alkaline-range analytical isoelectric focusing in thin-layer polyacrylamide gel: Application to cationic proteins in serum

Abstract

Two-stage methods for alkaline-range analytical isoelectric focusing in thin-layer polyacrylamide gels have been developed for the analysis of the cationic proteins in complex mixtures of protein. A pH gradient covering the “entire” pH range but emphasizing only a narrow portion of this gradient is produced. During the first stage a nonlinear pH gradient is nearly established. The anodic electrode is then moved to a position on the gel such that the applied potential is exclusively across the alkaline portion of the gradient. The second stage results in a significant increase in the electric field strength and, consequently, the resolving power. The two-stage methods, exploiting the advantages of narrow pH gradients and high field strengths, have enabled excellent resolution of the highly heterogeneous cationic proteins in serum.