The PhastSystem equipment used for crossed immunoelectrophoresis combined with immunoblotting of coprecipitated monoclonal antibodies as studied with platelet membrane receptor proteins

Abstract

The Pharmacia PhastSystem equipment has been used for crossed immunoelectro-phoresis combined with a technique for immunoblotting with monoclonal antibodies. This miniaturized gel system is compared to the conventional approach using platelet membrane receptor proteins as a model. Whereas in the conventional system the electrophoretic procedure takes place within 20 h, 3 h are adequate for the small gel system. Because of the short second-dimensional electrophoresis, and only one over-night incubation, the total electrophoretic and blotting procedure could be reduced from about 48 h to 24 h. The amount of antiserum used during the second-dimensional electrophoresis could be reduced roughly by a factor of 5. The examples with electrophoresis and immunoblotting using platelet extracts in 1 % Triton X-100 demonstrate that membrane receptor proteins can be studied even when present as noncovalent complexes. The immunoblotting can be used with monoclonal antibodies that do not function in Western blotting.