Onedimensional Polyacrylamide Gel Electrophoresis

P. G. Righetti, University of Verona, Verona,

Italy

Copyright © 2000 Academic Press

Electrophoresis is based on the differential migration of electrically charged particles in an electric field. As such, the method is applicable only to ionic or ionogenic materials, i.e. substances convertible to ionic species (a classic example being neutral sugars, which form negatively charged complexes with borate). In fact, with the advent of capillary zone electrophoresis (CZE) it has been found that a host of neutral substances can be induced to migrate in an electric field by inclusion in charged micelles, e.g. of anionic (sodium dodecyl sulfate, SDS) or cationic (cetyltrimethylammonium bromide, CTAB) surfactants. Even compounds that are not ionic, ionogenic, or complexable can often be analysed by CZE as they are transported past the detector by the strong elec-troosmotic flow on the capillary walls.

Basically, if one plots the velocity of a zone against the pH in the same zone, electrophoretic techniques can be divided into four main types: zone electrophoresis (ZE) together with moving-boundary electrophoresis (MBE), discontinuous (disc) electrophoresis, isotachophoresis (ITP) and isoelectric focusing (IEF). Figure 1 represents this classification. It can be seen that IEF and ITP are based on principles that are 'perpendicular' to ZE and MBE. In particular, in IEF, once steady-state conditions have been attained, all proteins reach a zero-velocity (v) thus they remain immobile (v = 0, pH-axis). It is then clear that ITP closes the ring of possibilities: all zones move with the same velocity, but at different pH. Alternatively, electrophoretic techniques may be enumerated in chronological order, as follows: moving boundary electrophoresis (MBE), zone electrophor-esis (ZE), disc electrophoresis, isoelectric focusing (IEF), sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional (2-D) maps, isotachophoresis (ITP), staining techniques, immobilized pH gradients (IPG), and capillary zone electrophoresis.

ZE became a reality when hydrophilic gels (acting as an anticonvective support) were discovered. Grabar and Williams in 1953 first proposed the use of an agar matrix (currently abandoned in favour of a highly purified agar fraction, agarose). They also combined, for the first time, electrophoresis on a hydrophilic support with biospecific detection (im-munoelectrophoresis). Barely two years after that, Smithies (1955) applied another gel, potato starch. The starch blocks were highly concentrated matrices (12-14% solids) and subsequently introduced a new parameter in electrophoretic separations: molecular sieving. Human sera, which in cellulose acetate or paper electrophoresis, were resolved in barely five bands, now produced a spectrum of 15 zones. The most important discovery, however, came with the introduction of polyacrylamide gels and disc elec-trophoresis; this discovery was thoroughly debated in

Figure 1 Classificationofthefourmodesofelectrokinetictech-niques. The velocity of a zone is plotted against the pH in the same zone. (A) zone and moving boundary electrophoresis; (B) discontinuous electrophoresis; (C) isoelectric focusing; (D) isotachophoresis. (Reproduced with permission from Routs RJ (1971) PhD thesis, University of Eindhoven.

Figure 1 Classificationofthefourmodesofelectrokinetictech-niques. The velocity of a zone is plotted against the pH in the same zone. (A) zone and moving boundary electrophoresis; (B) discontinuous electrophoresis; (C) isoelectric focusing; (D) isotachophoresis. (Reproduced with permission from Routs RJ (1971) PhD thesis, University of Eindhoven.

a classic electrophoretic volume, which appeared in December, 1964 in the Annals of the New York Academy of Sciences (a collectors item!). This was like the explosion of a supernova in the firmament of electrokinetic methodologies. Although most of the above-mentioned techniques belong to the category of one-dimensional PAGE, we will only mention three of them here in detail: disc electrophoresis, SDS electrophoresis and pore-gradient-gel electrophoresis. The other techniques such as IEF and ITP, being steady-state methods, are best performed in non-sieving media. The technique of moving-boundary electrophoresis died out long ago.

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