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In spite of the high sensitivity of silver staining and the wide dynamic range of various fluorescent detection methods, Coomassie Brilliant Blue staining is still the most widely used protein detection technique for proteins separated by polyacrylamide gel electrophoresis. There are several reasons: Low price, Visible with the eye, Desk top scanners can be employed for image acquisition, Better for quantitative analysis than silver staining, Possible modifications for fast or highly sensitive staining, Mass spectrometry compatible.

The study of the “proteomes” of human cells, tissues, and body fluids is a big challenge, and several highly sophisticated workflow approaches are pursued to achieve as comprehensive information as possible. Initially proteome analysis was exclusively based on the gel-based workflow, employing two-dimensional electrophoresis of protein extracts followed by mass spectrometry of the tryptic peptide digests of protein spots. Meanwhile several additional proteomics workflows are applied, which are mostly based on separation and analysis of tryptic peptides without separating the protein mixture. However, direct information on quantitative and qualitative changes of protein expressions can only be obtained by methods operating on the protein level, no other method can replace two-dimensional electrophoresis. In this review we compile the different techniques of high-resolution two-dimensional electrophoresis and their further developments to increase the degree of reliance of the method.

Publisher Summary Isoelectric focusing (IEF) is most commonly carried out in polyacrylamide slab gels. Over the years, the slab format has proved preferable to other possible configurations for the technique. The first practical realization of IEF was performed in glass columns containing sucrose density gradients. The pH gradients were generated with carrier ampholytes and the sucrose density gradient stabilized focused bands against precipitation. This chapter presents the first apparatus for performing IEF in polyacrylamide slab gels. The chapter describes the most successful approaches to slab gel IEF. The phenomenon called “electroendosmosis” can destroy a pH gradient and ruin resolution. It occurs when the separation matrix and (or) parts of the equipment that are in direct contact with the gel contain fixed charges. Polyacrylamide is an ideal matrix for IEF with both carrier ampholytes and immobilized pH gradients. It is chemically inert, compact, completely transparent, and free of electroendosmosis. Some laboratories use vertical slab gel electrophoresis equipment for IEF. In this case, the gel remains in the casting cassette between two glass plates during the run. There are two ways to polymerize polyacrylamide gels: (1) chemical polymerization with tetramethylethylenediamine (TEMED) and ammonium persulfate; and (2) photopolymerization with riboflavin and TEMED.

Using polyacrylamide electrode gels during horizontal sodium lauryl sulfate-polyacrylamide electrophoresis of radiolabeled proteins instead of electrode papers reaching into electrode buffer reservoirs, silver staining is improved and we reduce chemical and radioactive liquid waste during electrophoresis.

Buffer-soluble seed proteins of 12 varieties of winged bean (Psophocarpus tetragonolobus L.) were separated by ultrathin-layer isoelectric focusing (IEF) and sodium dodecylsulphate electrophoresis(SDS-PAGE). Whereas the molecular weight spectra of the proteins obtained by SDS-PAGE were identical for all varieties studied, the IEF patterns showed variety specificity for each sample. Most of the variety specific protein bands were identified as glycoproteins. The zymograms of the isoelectically focused trypsin inhibitors were variety specific in both activity and isoelectric point distribution.