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Protein (Western) Blotting with Immunodetection

  • John M. Walker
Protocol
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Protein blotting simply involves transferring separated protein bands from an acrylamide gel onto a more stable and immobilizing medium, such as nitrocellulose paper. Once transferred to the immobilizing medium, a variety of analytical procedures may be carried out on the proteins that would otherwise have proved difficult or impossible in the gel. Such procedures may include hybridization with labeled DNA or RNA probes, detection with antibodies (as used in this experiment), detection by specific staining procedures, autoradiographic assay, and so on. The approach used here is exactly analogous to the method used to transfer DNA from agarose gels (the Southern Blot, see Chapter 5) and has been given the name “Western” blotting (1, 2, 3). There are a number of advantages to working with a protein blot rather than the original gel. These include:
  1. 1.

    Rapid staining/destaining: once transferred, staining and destaining can be achieved within 5 min.

     
  2. 2.

    There is no ampholyte staining when analyzing IEF gels.

     
  3. 3.

    Samples in preparative gels may be located rapidly by briefly blotting the gel.

     
  4. 4.

    Low concentrations of samples can be detected since they are not spread throughout the thickness of a gel, but “concentrated” at the surface of the paper. As a consequence, it is possible to use lower specific activities and lower concentrations of expensive or scarce probes.

     
  5. 5.

    The “blot” is a convenient permanent record of the gel run.

     

Keywords

Scalpel Blade Lower Specific Activity Protein Blotting Nitrocellulose Sheet Nitrocellulose Paper 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Burnett, W. (1981) ‘Western blotting’: Electrophoretic transfer of proteins from sodium dodecyl sulfate polyacrylamide gels to unmodified nitrocellulose. Anal. Biochem. 112, 195–203.CrossRefGoogle Scholar
  2. 2.
    Towlin, H., Stachelin, 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(9), 4350–4354.CrossRefGoogle Scholar
  3. 3.
    Bittner, M., Kupfer, P., and Morris, C.F. (1980) Electrophoretic transfer of proteins and nucleic acids from slab gels to diazobenzyloxymethyl cellulose or nitrocellulose sheets. Anal Biochem. 102, 459–471.PubMedCrossRefGoogle Scholar

Further Reading

  1. Vaessen, R.T.M.J., Kreike, J., and Groot, G.S.P. (1981) Protein transfer to nitrocellulose filters. FEBS Let. 124, 193–196.CrossRefGoogle Scholar
  2. Lin, W. and Kasamatsu, H. (1983) On the electrotransfer of polypeptides from gels to nitrocellulose membranes. Anal. Biochem. 128, 302–311.PubMedCrossRefGoogle Scholar
  3. Notes on Immunoblotting. A free eight-page booklet, Mercia Brocades, Surrey, England.Google Scholar

Copyright information

© The Humana Press Inc. 1986

Authors and Affiliations

  • John M. Walker
    • 1
  1. 1.Division of Biological and Environmental studiesThe Hatfield PolytechnicUK

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