Western Blotting: Remembrance of Things Past

  • W. Neal Burnette
Part of the Methods in Molecular Biology book series (MIMB, volume 536)


Western blotting sprung from the need to develop a sensitive visual assay for the antigen specificity of monoclonal antibodies. The technique employed SDS-PAGE of protein antigens, electrophoretic replica transfer of gel-resolved proteins to unmodified nitrocellulose sheets, probing the immobilized antigens with hybridomas, and detection of antibody–antigen complexes with radiolabeled staphylococcal protein A and autoradiography. The simplicity and relevance of the method has led to its expansive application as an immunodiagnostic and a ubiquitous research tool in biology and medicine.

Key words

Western blotting SDS-PAGE Electroblotting Unmodified nitrocellulose Immobilized replica Antigen specificity Antibody–antigen complex Protein A 


  1. 1.
    Swift, J. (1726) Travels into Several Remote Nations of the World, in Four Parts. By Lemuel Gulliver, First a Surgeon, and then a Captain of several Ships. Benjamin Motte, London.Google Scholar
  2. 2.
    Burnette, W.N. (1981) “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 112, 195–203.PubMedCrossRefGoogle Scholar
  3. 3.
    Köhler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497.PubMedCrossRefGoogle Scholar
  4. 4.
    Nowinski, R.C., Stone, M.R., Tam, M., Lostrom, M.E., Burnette, W.N., et al (1980) Mapping of viral proteins with monoclonal antibodies: analysis of the envelope proteins of murine leukemia viruses, in Monoclonal Antibodies: Hybridomas: A New Dimension in Biological Analyses (Kennett, R.H., McKearn, T.J., and Bechtol, K.B., eds.), Plenum, New York, pp. 295–316.Google Scholar
  5. 5.
    Langone, J.J. (1978) [125I]protein A: a tracer for general use in immunoassay. J Immunol Methods 24, 269–285.PubMedCrossRefGoogle Scholar
  6. 6.
    Alwine, J.C., Kemp, D.J., and Stark, G.R. (1977) Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci USA 74, 5350–5354.PubMedCrossRefGoogle Scholar
  7. 7.
    Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76, 4350–4354.PubMedCrossRefGoogle Scholar
  8. 8.
    Renart J., Reiser, J., and Stark, G.R. (1979) 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 USA 76, 3116–3120.PubMedCrossRefGoogle Scholar
  9. 9.
    Burnette, W.N. (1991) Western blotting (Citation Classic). Curr Contents 34, 8.Google Scholar
  10. 10.
    Campanario, J.M. (1996) Have referees rejected some of the most-cited articles of all times? J Am Soc Inf Sci 47, 302–310.CrossRefGoogle Scholar
  11. 11.
    Proust, M. (1913) À la recherche du temps perdu (vol. I). Bernard Grasset, Paris.Google Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Molecular Pharmaceutics CorporationChapel HillUSA

Personalised recommendations