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Two-dimensional electrophoresis as a complementary approach to gel-free proteomic methods possesses the ability to separate physiologically important isoforms of proteins in an unbiased manner. Frequently, those isoforms are low-abundant regulators, and therefore, detection and identification of low-abundant proteins is highly necessary to exploit this advantage. We describe an experimental sequence of classical operations to process gels but optimized them, in order to identify each detectable protein spot on gel.

Separating proteins according to two different gel-electrophoretic methods not only increases the resolution power for highly complex samples when compared to one-dimensional separations, but is also a valuable tool for protein and protein complex characterization. There are a number of different electrophoresis methods which can be combined. The combination of isoelectric focusing under denaturing conditions and SDS polyacrylamide gel electrophoresis delivers the highest resolution of all bio-analytic techniques. This is a short review on the history and state of the art of two-dimensional electrophoresis methods, and contains some practical tips for high resolution 2D electrophoresis, which are based on several decades of experience with this method.

Extremely wide immobilized pH gradients, pH 2.5-11, for isoelectric separation of complex protein mixtures are described. These pH gradients are theoretically and practically the maximum that can be achieved at present with the available acrylamido buffers and titrants. Conditions are described for reducing conductivity and electroendosmosis in extreme pH ranges. Furthermore, new conditions are described for the separation of proteins in the second dimension. Using this protocol, nearly all the possible cellular products can be separated in one single two-dimensional map.

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.

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.

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.

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.

A new method for electrophoretic retrieval of protein zones from Immobiline matrices is described, based on elution directly in a free liquid phase, rather than in ion-exchange beads or molecular sieves, as previously described. The chopped Immobiline gel is loaded on top of a 5% T stacking gel, 6-10 mm in height, and forced to transverse it and collect into a chamber, filled with 20% sucrose solution, closed on its anodic side by a dialysis sac. The transfer is practically quantitative, for most proteins, after 30-60 min of zone electrophoresis at 10 W (300 V potential differential). Recovery of protein mass is in general better than 90%, while for enzyme activity is in the range of 60-80%. For preserving enzyme integrity, the following precautions are recommended: short electrophoretic times; avoidance of anodic oxidation; chilling of the buffer in the anodic chamber; and use of low levels (2-5 mM) of the specific enzyme substrate throughout the entire electrophoretic system (cathode, anode and gel plug).

A method of ultrathin-layer isoelectric focusing in 0.12-, 0.24-, or 0.36-mm polyacrylamide gel layers polymerized on a sheet of cellophane as support is deseribed. The gel adheres firmly to the cellophane during all operation steps, is protected from fracture, and can be handied very conveniently. Resolution is markedly improved in ultrathin gels in comparison with the conventional 1- to 2-mm-thick gels. Staining and destaining are completed in a substantially shorter time than so far achieved. The ultrathin gels can be easily dried on the cellophane, a perfectly transparent record being obtained for future reference and for densitometric evaluation. Results are presented for a number of commercial proteins and legume seed proteins. The advantages of ultrathin-layer isoelectric focusing are discussed.

A new technique for generating pH gradients in isoelectric focusing is described, based on the principle that the buffering groups are covalently linked to the matrix used as anticonvective medium. For the generation of this type of pH gradient in polyacrylamide gels, a set of buffering monomers, called Immobiline (in analogy with Ampholine), is used. The pH gradient gels are cast in the same way as pore gradient gels, but instead of varying the acrylamide content, the light and heavy solutions are adjusted to different pH values with the aid of the Immobiline buffers. Available Immobiline species make it possible to generate any narrow linear pH gradient between pH 3 and 10. The behaviour of these types of gradients in isoelectric focusing is described. Immobilized pH gradients show a number of advantages compared with carrier ampholyte generated pH gradients. The most important are: (1) the cathodic drift is completely abolished; (2) they give higher resolution and higher loading capacity; (3) they have uniform conductivity and buffering capacity; (4) they represent a milieu of known and controlled ionic strength.

The human serum transferrin (Tf) system was analyzed by isoelectric focusing (IEF) with immobilized pH gradients. For the demonstration of the genetic variability the Fe1-Tf region was chosen. The pH range suitable for analysis of the Tf system was pH 5.20 to pH 5.75. The phenotypes of the common six TfC subtypes are described. No further heterogeneity among TfC1, TfC2, and TfC3 was noted. Also presented are the phenotypes of the TfC6 subtype, and of three different TfB and three different TfD variants. IEF with immobilized pH gradients appears to be a suitable method for the analysis of the inherited transferrin polymorphism.