Effects of the blocking agents bovine serum albumin and Tween 20 in different buffers on immunoblotting of brain proteins and marker proteins.
The effects of the blocking agents bovine serum albumin and Tween 20 in buffers at pH values 7.2 and 10.2 were compared in immunoblotting with 2 different antisera. The antisera were raised against a purified brain-specific protein fraction from human brain, soluble in perchloric acid, and phosphate-activated glutaminase from pig brain, respectively. The antigens were a crude perchloric acid-soluble brain extract, a crude brain phosphate-activated glutaminase fraction, and proteins commonly used as molecular weight markers. The binding patterns of the 2 antisera to the respective brain antigen preparations changed, depending on the blocking agent and the pH of the blocking buffer. Also, antibody binding to the molecular weight marker proteins was observed with some of the blocking buffers. Immunoblotting with Tris-saline, pH 10.2, containing 3% bovine serum albumin as blocking agent and diluting buffer for the antisera, showed negligible antibody binding to the marker proteins and most specific binding to the brain antigens.
D001921:Brain / Q000276:immunology*; D001923:Brain Chemistry; D005972:Glutaminase / Q000276:immunology; D006801:Humans; D007163:Immunosorbent Techniques; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000276:immunology*; D011136:Polysorbates; D012710:Serum Albumin, Bovine / Q000276:immunology*
D009419:Nerve Tissue Proteins; D011136:Polysorbates; D012710:Serum Albumin, Bovine; D005972:Glutaminase