DISC ELECTROPHORESIS   I

Although electrophoresis is one of the most effective methods for the separation of ionic components of a mixture, the resolving power of different electrophoretic methods is quite variable. To separate two component ions, it is necessary to permit migration to continue until one of the kinds of ions has traveled at least one thickness of the volumes that it initially occupied (the starting zone) further than the other. However, the sharpness, and therefore the resolution, of the zones occupied by each ion diminishes with time because of the spreading of the zones as a result of diffusion. Remarkable resolution has been achieved when advantage is taken of the frictional properties of gels to aid separation by seiving at the molecular level (see Smithies^l). A new method, disc electrophoresis,Ý has been designed that takes advantage of the adjustability of the pore size of a synthetic gel and that automatically produces starting zones of the order of 10 microns thickness from initial volumes with thicknesses of the order of centimeters. High resolution is thus achieved in very brief runs.

With this technique, over 20 serum proteins are routinely separated from a sample of whole human serum as small as one microliter in a 20-minute run (see FIGURE 1). Direct analysis of even very dilute samples becomes routine because the various ions are automatically concentrated to fixed high values at the beginning of the run just prior to separation. Preliminary laboratory studies and theoretic considerations provide evidence of the applicability of this technique to a wide range of ionic species for both analytic and large-scale preparative purposes.

Theory has also provided the basis for a simple application of disc electrophoresis to the simultaneous determination of both the free mobility and the aqueous diffusion constant of a protein.

This report will detail some mechanisms that provide a rationale for the resolution afforded by zone electrophoresis in many gels; will develop the theory of some new modifications of zone electrophoresis that have been designed to take maximum advantage of these mechanisms; and will provide some examples of the results that disc electrophoresis has produced.

This study was first stimulated by the revolutionary results of the starch gel technique of Smithies² and the application of this technique by Hunter^3,4 for producing "zymograms" of enzymes of histochemical interest. Our recent cytochemical studies,^5-7 which were then well under way, promised to be substantially clarified by the application of Hunter's technique.⁸ We had heard of the variability of the starch gel and of difficulties in its preparation. We also had had

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* The present manuscript is an expanded and updated version of a paper that was first made available to the scientific public in preprmt form in January, 1962, through the generosity of the Distillation Products Division of Eastman Kodak Company, Rochester, N. Y.

Ý The name was derived from the dependence of the new technique on discontinuities in the electrophoretic matrix and, coincidentally, from the discoid shape of the separated zones of ions in the standard form of our technique.