HIGH-RESOLUTION SLAB GEL ISOELECTRIC-FOCUSING - METHODS FOR QUANTITATIVE ELECTROPHORETIC TRANSFER AND IMMUNODETECTION OF PROTEINS AS APPLIED TO THE STUDY OF THE MULTIPLE ISOELECTRIC FORMS OF ORNITHINE DECARBOXYLASE

A high-resolution isoelectric focusing vertical slab gel method which can resolve proteins which differ by a single charge was developed and this method was applied to the study of the multiple isoelectric form s of ornithine decarboxylase. Separation of proteins at this high leve l of resolution was achieved by increasing the ampholyte concentration in the gels to 6%. Various lots of ampholytes, from the same or diffe rent commercial sources, differed significantly in their protein bindi ng capacity. Ampholytes bound to proteins interfered both with the ele ctrophoretic transfer of proteins from the gel to immunoblotting membr anes and with the ability of antibodies to interact with proteins on t he immunoblotting membranes. Increasing the amount of protein loaded i nto a gel lane also decreased the efficiency of the electrophoretic tr ansfer and immunodetection. To overcome these problems, both gel washi ng and gel electrophoretic transfer protocols for disrupting the ampho lyte-protein binding and enabling a quantitative electrophoretic trans fer of proteins were developed. Two gel washing procedures, with eithe r thiocyanate or borate buffers, and a two-step electrophoretic transf er method are described. The choice of which method to use to optimall y disrupt the ampholyte-protein binding was found to vary with each lo t of ampholytes employed. (C) 1994 Academic Press, Inc.