Disc Electrophoresis. Ii. Method and Application to Human Serum Proteins.

The purpose of this paper is to describe a technique of zone electrophoresis in polyacrylamide gels. The salient characteristics of the technique reside in (1) the controlled variation of the gel pore size for the purpose of increasing the resolution of ions based on dimensional differences and (2) an electrophoretic step for concentrating the sample ions into a narrow starting zone prior to electrophoretic separation. Zone electrophoresis provides a comparatively innocuous method for the separation of ionic mixtures and is based on differences among the electrophoretic mobilities of the constituent ions. In most zone techniques separation is dependent solely on the differences in the free electrophoretic mobilities among the constituent ions, and the supporting matrix, e.g., filter paper, is used primarily to reduce convection. In those instances when the differences among the free mobilities are small, satisfactory separation may not be achieved. With the development of zone electrophoresis in starch gel matrices, the capacity of gels to "sieve" high molecular weight substances such as proteins was recognized to provide a simple but elegant means to resolve ions of similar, and even identical, free mobilities.l A gel matrix, unlike other porous media such as filter paper or starch granules, is a latticed structure with pores of molecular dimensions that can impose an appreciable frictional resistance to the passage of ions, provided the size of the pores approaches the dimensions of the migrating ions. If, as is the case with starch gels,2 the average pore size approaches the range of dimensions of proteins, the various protein fractions will be differentially retarded to degrees proportional to their dimensions. Thus the separation of mixtures is achieved in such a gel matrix through dimensional as well as charge differences. The substantial increase in resolving power of sieving gel matrices over that of conventional filter paper electrophoresis is effectively demonstrated with a complex protein mixture such as human serum. In a single electrophoretic analysis (pH 8.6) a filter paper technique commonly resolves five to seven protein fractions, while starch gel resolves 20 to 30 fractions. This discovery by Smithiesl,2 of the additional resolving power provided by the sieving capacity of gels prompted our investigation of polyacrylamide gels. Polyacrylamide gels are synthetic polymers formed from low molecular weight chemicals obtainable in high purity. The pore size of polyacrylamide gels can be varied through a wide range by adjustment of the monomer concentration, and preparation of the gels is a simple and rapid procedure. Furthermore, these gels are transparent to visible radiation through a wide range of monomer