Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1983;2(2):233–237. doi: 10.1002/j.1460-2075.1983.tb01411.x

Antibody-linked polymerase assay on protein blots: a novel method for identifying polymerases following SDS-polyacrylamide gel electrophoresis.

J van der Meer 1, L Dorssers 1, P Zabel 1
PMCID: PMC555119  PMID: 11894932

Abstract

We describe a method for correlating polymerase activity with a particular polypeptide band in an SDS-polyacrylamide gel which does not require renaturation of the SDS-denatured enzyme. The method involves the following steps: (i) transfer of proteins from an SDS-polyacrylamide gel onto nitrocellulose; (ii) incubation with excess antiserum raised against a partially purified polymerase preparation to link one Fab site of an antibody molecule to the denatured enzyme on the nitrocellulose; (iii) binding of native polymerase to the other Fab site of the antibody molecule in the immune complex to generate a specific polymerase 'sandwich'; (iv) assaying of the nitrocellulose filter for antibody-linked native polymerase activity using an appropriate template and a radioactive substrate followed by treatment with trichloroacetic acid to precipitate in situ the radioactive product. The essential feature of this method is that the use of both non-specific anti-polymerase serum and a partially purified enzyme preparation is sufficient to allow identification of a specific protein following SDS-polyacrylamide gel electrophoresis. This antibody-linked polymerase assay has been developed to identify a 130,000-dalton RNA-dependent RNA polymerase from cowpea leaves. Possible applications of this type of assay as a tool for identifying a wide variety of proteins are discussed.

Full text

PDF
233

Images in this article

234Fig. 1.
on p.234
234Fig. 2.
on p.234
235Fig. 3.
on p.235

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bowen B., Steinberg J., Laemmli U. K., Weintraub H. The detection of DNA-binding proteins by protein blotting. Nucleic Acids Res. 1980 Jan 11;8(1):1–20. doi: 10.1093/nar/8.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  3. Chang L. M., Plevani P., Bollum F. J. Evolutionary conservation of DNA polymerase beta structure. Proc Natl Acad Sci U S A. 1982 Feb;79(3):758–761. doi: 10.1073/pnas.79.3.758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Hager D. A., Burgess R. R. Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal Biochem. 1980 Nov 15;109(1):76–86. doi: 10.1016/0003-2697(80)90013-5. [DOI] [PubMed] [Google Scholar]
  5. Huet J., Sentenac A., Fromageot P. Detection of nucleases degrading double helical RNA and of nucleic acid-binding proteins following SDS-gel electrophoresis. FEBS Lett. 1978 Oct 1;94(1):28–32. doi: 10.1016/0014-5793(78)80899-0. [DOI] [PubMed] [Google Scholar]
  6. Lacks S. A., Springhorn S. S. Renaturation of enzymes after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. J Biol Chem. 1980 Aug 10;255(15):7467–7473. [PubMed] [Google Scholar]
  7. Muilerman H. G., ter Hart H. G., Van Dijk W. Specific detection of inactive enzyme protein after polyacrylamide gel electrophoresis by a new enzyme-immunoassay method using unspecific antiserum and partially purified active enzyme: application to rat liver phosphodiesterase I. Anal Biochem. 1982 Feb;120(1):46–51. doi: 10.1016/0003-2697(82)90315-3. [DOI] [PubMed] [Google Scholar]
  8. Renart J., Reiser J., Stark G. R. Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3116–3120. doi: 10.1073/pnas.76.7.3116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Rosenthal A. L., Lacks S. A. Detection of histones by DNA binding ability after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. J Biol Chem. 1978 Dec 25;253(24):8674–8676. [PubMed] [Google Scholar]
  10. Rosenthal A. L., Lacks S. A. Nuclease detection in SDS-polyacrylamide gel electrophoresis. Anal Biochem. 1977 May 15;80(1):76–90. doi: 10.1016/0003-2697(77)90627-3. [DOI] [PubMed] [Google Scholar]
  11. Spanos A., Sedgwick S. G., Yarranton G. T., Hübscher U., Banks G. R. Detection of the catalytic activities of DNA polymerases and their associated exonucleases following SDS-polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Apr 24;9(8):1825–1839. doi: 10.1093/nar/9.8.1825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Symington J., Green M., Brackmann K. Immunoautoradiographic detection of proteins after electrophoretic transfer from gels to diazo-paper: analysis of adenovirus encoded proteins. Proc Natl Acad Sci U S A. 1981 Jan;78(1):177–181. doi: 10.1073/pnas.78.1.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Zabel P., Jongen-Neven I., Van Krammen A. In vitro replication of cowpea mosaic virus RNA. II. Solubilization of membrane-bound replicase and the partial purification of the solubilized enzyme. J Virol. 1976 Mar;17(3):679–685. doi: 10.1128/jvi.17.3.679-685.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Zabel P., Moerman M., van Straaten F., Goldbach R., van Kammen A. Antibodies Against the Genome-Linked Protein VPg of Cowpea Mosaic Virus Recognize a 60,000-Dalton Precursor Polypeptide. J Virol. 1982 Mar;41(3):1083–1088. doi: 10.1128/jvi.41.3.1083-1088.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES