TRANSFER OF PROTEINS FROM GELS TO DIAZOBENZYLOXYMETHYL-PAPER AND DETECTION WITH ANTISERA - METHOD FOR STUDYING ANTIBODY SPECIFICITY AND ANTIGEN STRUCTURE

The authors describe a rapid and very sensitive method for detecting proteins as antigens after their separation in polyacrylamide/agarose composite gels, with or without sodium dodecyl sulfate. The polyacrylamide matrix is crosslinked with a reagent that can be cleaved with periodate or alkali to facilitate transfer of the protein bands to diazobenzyloxymethyl-paper, where they are coupled covalently. Specific proteins are detected by autoradiography after sequential incubation with unfractionated, unlabeled specific antiserum and 125I-labeled protein A from Staphylococcus aureus. Antibody and protein A can be removed with urea and 2-mercaptoethanol, and the same paper can be probed again with a different antiserum. An antiserum specific for the simian virus 40 virion proteins VP3 and VP2 has been prepared; it does not crossreact with VP1, as demonstrated by this method. An antiserum raised in rabbits against simian virus 40-transformed rabbit kidney cells is shown to be directed primarily against a periodate-sensitive moiety present in tumor (T) antigen from infected or transformed cells, whereas an antiserum raised in rabbits against large T antigen purified from lytically infected monkey kidney cells by electrophoresis in the presence of sodium dodecyl sulfate is directed primarily against determinants that are not sensitive to periodate.