ACRYLAMIDE GEL ELECTROPHORESIS

This symposium on gel electrophoresis includes in its program papers on starch gel electrophoresis, agar gel electrophoresis, acrylamide gel electrophoresis, and the specialized technique of disc electrophoresis. It is appropriate to consider what characteristic distinguishes gel electrophoresis from other techniques and makes it especially useful in the analysis of protein and other complex mixtures. In my opinion it is the “sieve” or “molecular filtration” effect, first described by 0. Smithies,’ that is the essential characteristic of gel electrophoresis. The combination of electrophoretic effects and molecular filtration effects produces new and startling resolutions that cannot be obtained by other electrophoretic procedures. This combination‘of effects is not present in agar gel electrophoresis. Therefore agar techniques should logically be classified with paper electrophoresis techniques, which they most closely resemble with respect to the type of pattern produced. The specialized technique of “disc electrophoresis” intrinsically applies a discontinuous buffer system to sharpen the electrophoretic zones of the individual components of a mixture but does not in itself make use of the special feature of gel electrophoresis, which is the molecular filtration effect. Immunoelectrophoresis is also a specialized technique, namely a technique of detecting and identifying the zones separated by electrophoresis. It could equally well be applied to a paper electrophoresis type of separation, as it is in agar gel, or to a molecular filtration type of separation as is produced by starch gel or acrylamide gel. These considerations provide a rational basis for focusing our attention on the special problems that have arisen in gel electrophoresis (defined as electrophoresis technique combining both electrophoretic and molecular filtration effects) and the special results available from it. It is, however, beyond the scope of this paper to consider the theoretical problems of electrophoresis as modified by the filtration effect. Instead, this paper is limited to the applications of gel electrophoresis and to the problems we have encountered with it since our first publication in 1959.2 It is evident from a quick perusal of the program that we are devoting most of our attention to the disc electrophoresis technique of Ornstein. Since, apparently, I am the only speaker to describe the vertical slab technique I must begin with a description of our procedure and apparatus. Our procedure starts with a vertical gel slab prepared in a specially designed cell (FIGURE combining gel mold, buffer reservoirs and electrodes, and cooling plates all in one unit. We prefer a flat slab in contrast to a cylindrical gel for several reasons. First, the flat slab provides maximum surface area for cooling the gel. Second, the resulting patterns are easier to quantitate in standard recording densitometers. Third, a large number of samples can be processed in a single gel, making the technique easier to carry out in the laboratory and facilitating the direct intercomparison of specimens processed under identical conditions all the way through to the final densitometer recording. Fourth, and most important, the flat slab permits the application of two-dimensional techniques, which is impossible in the vertical tube apparatus. We have never observed any difficulties with stress relief or separation from