Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure.
We 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 [Lane, D.P. & Robbins, A.K. (1978) Virology 87, 182-193] is directed primarily against determinants that are not sensitive to periodate.
D000918:Antibody Specificity; D000956:Antigens, Viral / Q000302:isolation & purification; D001345:Autoradiography; D003979:Diazonium Compounds; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D007202:Indicators and Reagents; D011506:Proteins / Q000276:immunology* / Q000302:isolation & purification; D013539:Simian virus 40 / Q000276:immunology; D014764:Viral Proteins / Q000276:immunology
D000956:Antigens, Viral; D003979:Diazonium Compounds; D000939:Epitopes; D007202:Indicators and Reagents; D011506:Proteins; D014764:Viral Proteins; C015794:diazobenzyloxymethyl groups