Coomassie brilliant blue R-250 as a highly sensitive pre-stain for immediate visualization of human serum proteins on polyacrylamide gel disc electrophoresis and raising monospecific antibodies.

We hereby describe electrophoretic analysis of normal human serum pre-stained with Coomassie Brilliant Blue R-250 (CBB). The proposed method was optimized by studying as many as 450 disc electrophoretic separations and 30 variables. The method when compared with the post-electrophoretic staining by Amido Black (AB) revealed that the pre-stained discs were intensely well defined and resolved within 2 hours with a transparent gel. Gels stained with AB, despite a prolonged destaining, showed a residual dye retention making identification of the faint components difficult. Protein eluted from the CBB pre-stained gels retained its purity and immunoreactivity and conjugates of the two prototype proteins namely the albumin and the transferrin produced high-titre monospecific antisera in immunized rabbits.

Identification of C-3 on polyacrylamide gel by pre-staining the normal human serum with Remazol Brilliant Blue and raising monospecific anti C-3 antibody.

Based on our earlier work we hereby describe pre-staining of normal human serum with Remazol Brilliant Blue (RBB) to precisely locate the third component of complement system (C3,B1C) on polyacrylamide gels. Testing the dye-protein complex eluted from different immunoreactive post-transferrin (origin to transferrin) components against the standards anti-C3 antibody in various gel precipitation techniques, revealed that the prominent band just posterior to the centrally placed transferrin is of complement. Subsequent to its identity, we also evolved a method to raise an anti-C3 antibody. The resulting antibody was found to be monospecific when assessed in different gel precipitation procedures.