High-Resolution Electrophoretic Purification and Structural Microanalysis of Peptides and Proteins

Abstract

Publisher Summary This chapter discusses the high-resolution electrophoretic purification and structural microanalysis of peptides and proteins. Traditional methods of protein purification have relied upon the physicochemical properties of the protein of interest, often taking advantage of one or more unique characteristics that can be exploited to remove unwanted contaminants. Several techniques being applied to achieve structural analyses are the practice of protein purification by high-resolution two-dimensional polyacrylamide gel electrophoresis (PAGE), electrotransfer to a chemically inert membrane, and high-sensitivity amino acid sequence determination. Until recently, the manipulation of the small amounts of proteins resolved on two-dimensional gels without serious losses restrained this procedure as a suitable alternative to existing purification techniques. The chapter provides historical perspectives of the developments of sodium dodecyl sulfate (SDS)-PAGE, protein electroblotting, and direct amino acid microsequencing, with special emphasis on those qualities that led to the evolution of a unified procedure for routine isolation and primary structural determination of proteins at the subnanomole level.