Quantitative electrophoretic transfer of polypeptides from SDS polyacrylamide gels to nitrocellulose sheets: a method for their re-use in immunoautoradiographic detection of antigens.
Polypeptides (233 micrograms) from brain synaptosomes resolved in SDS 7.5%--15% polyacrylamide gradient gels were electrophoretically transferred quantitatively to nitrocellulose sheets at 3 V/cm for 21 h in the following buffer: 25 mM Tris-192 mM glycine, pH 8.3/20% methanol/0.1% SDS. After immunoautoradiographic detection of antigens on this nitrocellulose replica with either rabbit anti-rat cerebrum immunoglobulins or a mouse monoclonal antibody to a rat synaptosomal protein, the antibody and [125I]protein A were removed from the replica by treatment with 8 M urea, 0.1 M 2-mercaptoethanol, and 5 mg/ml BSA at 60 degrees C for 1 h. The replicas were successfully re-used by re-exposing them to the antibody and [125I]protein A.
D000818:Animals; D000941:Antigens / Q000276:immunology*; D001345:Autoradiography; D001666:Binding Sites, Antibody; D001921:Brain / Q000276:immunology*; D002482:Cellulose / Q000031:analogs & derivatives; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000276:immunology; D010455:Peptides / Q000276:immunology*; D011817:Rabbits; D051381:Rats; D013205:Staphylococcal Protein A / Q000378:metabolism; D013574:Synaptosomes / Q000276:immunology
D000941:Antigens; D009419:Nerve Tissue Proteins; D010455:Peptides; D013205:Staphylococcal Protein A; D002482:Cellulose