Electroblotting of proteins from polyacrylamide gels for chemical characterization.

Abstract

Techniques of polyacrylamide gel electrophoresis (PAGE) have an almost unrivaled capacity for the separation of complex protein mixtures. In particular, two-dimensional methods (2-DE) can routinely separate up to 2000 proteins from whole cell and tissue homogenates, and using large format gel separations of up to 10,000 proteins have been described. Until recently, PAGE methods were used almost exclusively as analytical tools for the characterization of proteins by their physicochemical properties (charge, size, hydrophobicity) and relative abundance. However, the development of a battery of highly sensitive techniques of microchemical characterization, including N-terminal and internal protein microsequencing, amino acid compositional analysis, pep-tide mass profiling, and mass spectrometry (, ) has resulted in PAGE becoming the method of choice for the micro-preparative purification of protein for subsequent chemical analysis. These methods are described in detail in the relevant chapters in the present volume.