DENSITY GRADIENT ISOELECTRIC-FOCUSING OF PROTEINS IN ARTIFICIAL PH GRADIENTS MADE UP OF BINARY-MIXTURES OF AMPHOTERIC BUFFERS

A density gradient electrophoresis apparatus made of Perspex (7 cm, em pty set 2.2 cm) with a circular platinum anode and a palladium cathode was used for the separation of proteins in free liquid. Following a c oncept developed by M. Bier et al. (Electrophoresis 1993, 14, 1011-101 8), mixtures of two suitable amphoteric buffers I and II provide for m edia with a fixed and electrophoretically stable pH or were used for t he generation of preformed (electrophoretically stable) pH gradients c overing about 1 pH unit. Amphoters I and II are considered suitable if there is overlap between (pK(1,1)-1-2) and the pK(2,II)+1+2) region. 3-(N-Morpholino)propanesulfonic acid (MOPS) and gamma-amino-n-butyric acid (GABA) were used as an example. Two approaches were followed: (i) rate-zonal separation of test proteins in a pH window, formed by a fi xed ratio of MOPS/GABA. (ii) Isoelectric focusing in a shallow preform ed pH gradient, made up of inverse reciprocal linear gradients of MOPS and GABA. At isopH, test :proteins (bovine serum albumin, cytochrome c, ferritin, hemoglobin, lactoglobulin, myoglobin, and transferrin) we re rate-zonally separated within a short time. Even the separation of the A and B forms of lactoglobulin was feasible at isopH. The glycofor ms of transferrin were separated and enriched on a pH 5.2-6.1 pH gradi ent, indicating that pH differences of about 0.01 still permit resolut ion. Contrary to the ill-defined Ampholines, the cost of these well-de fined amphoters is low.