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By far the highest resolution of all separation techniques for intact proteins in a single analytical run continues to be by the combination of isoelectric focusing (IEF) and SDS-PAGE, originally introduced by O'Farrell [(1975). J. Biol. Chem.250, 4007-4021]. This analytical platform has seen a number of significant advances and applications over the past 25years, including reproducibility using immobilized pH gradient (IPG) strips [Bjellqvist et al. (1982). J. Biochem. Biophys. Methods6, 317-339.], resolution in alkaline IEF using hydroxyethyldisulfide (HED) [Olsson et al. (2002). Proteomics2, 1630-1632], and quantification for differential expression proteomics on intact proteins on a global scale [DIGE; Unlu et al. (1997). Electrophoresis18, 2071-2077]. These major improvements will be highlighted in this chapter alongside the principle and theory of 2D gel electrophoresis, as well as detailed methods for general 2D gel electrophoresis best use protocols.

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.