Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure.

TLDR

The study presents a rapid, highly sensitive method for detecting proteins separated in polyacrylamide/agarose gels, with or without SDS. The method uses a periodate‑cleavable crosslinked polyacrylamide matrix to transfer protein bands to diazobenzyloxymethyl paper, where they are covalently coupled and detected by autoradiography after sequential incubation with antiserum and 125I‑protein A, with the ability to strip and reprobe the paper. Using this method, an antiserum specific for SV40 VP3/VP2 was shown not to cross‑react with VP1, and rabbit antisera against SV40‑transformed kidney cells targeted a periodate‑sensitive tumor antigen moiety, whereas antisera against purified large T antigen targeted periodate‑insensitive determinants.

Abstract

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.