Advertisement

Detection of Proteins on Western Blots Using Chemifluorescence

  • Catherine Copse
  • Susan J. Fowler
Protocol
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Immunodetection of specific proteins that have been immobilized on membrane supports by Western blotting is a widely used protein analysis technique. Traditionally, radioactively labeled antibodies or ligands (usually 125I) were used to probe for specific antigens (1-3). Autoradiographic detection on X-ray film, for example, reveals the location of the antigen with reproducibly high sensitivity and the results are easily quantifiable. However, the need for lengthy exposure times and concerns related to the handling and disposal of radioactive materials have led to the development of nonradio-active techniques for Western blot analysis, for example, using colorimetric and chemi-luminescent detection.

Keywords

Amersham Pharmacia Biotech Charge Couple Device Camera Fluorescent Product Fluorescence Scanner Plastic Wrap 
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.
    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
  2. 2.
    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
  3. 3.
    Bowen, B., Steinberg, J., Laemmli, U. K., and Weintraub, H. (1980) The detection of DNA-binding proteins by protein blotting. Nucleic Acids Res. 8, 1–20.PubMedCrossRefGoogle Scholar
  4. 4.
    Gingrich, J. C., Davis, D. R., and Nguyen, Q. (2000) Multiplex detection and quantitation of proteins on Western blots using fluorescent probes. BioTechniques 29, 636–642.PubMedGoogle Scholar
  5. 5.
    Fradelizi, J., Friederich, E., Beckerle, M. C., and Golsteyn, R. M. (1999) Quantitative measurement of proteins by Western blotting with CyTM5-coupled secondary antibodies. BioTechniques 26, 484–494.PubMedGoogle Scholar
  6. 6.
    Akhavan-Tafti, H., DeSilva, R., Arghavani, Z., Eikholt, R. A., Handley, S., Schoenfelner, B. A., et al. (1998) Characterization of acridancarboxylic acid derivatives of chemilumines-cent peroxidase substrates. J. Org. Chem. 63, 930–937.CrossRefGoogle Scholar
  7. 7.
    Appendix 2, Fluorescence Imaging: Principles and Methods (2000) Amersham Pharmacia Biotech, publisher, state, pp. xx–xxGoogle Scholar
  8. 8.
    Kaufman, S., Ewing, C., and Shaper, J. (1987) The erasable Western blot. Analyt. Biochem. 161, 89–95.CrossRefGoogle Scholar
  9. 9.
    Van Dam, A. (1994) Transfer and blocking conditions in immunoblotting, in Protein Blotting-A Practical Approach (Dunbar, B. S., ed.), Oxford University Press, New York, pp. 73–85.Google Scholar
  10. 10.
    Mansfield, M. A. (1994) Transfer and blocking conditions in immunoblotting, in Protein Blotting-A Practical Approach (Dunbar, B. S., ed.), Oxford University Press, New YorkGoogle Scholar
  11. 11.
    Hoffman, W. L and Jump, A. A. (1989) Inhibition of the streptavidin-biotin interaction by milk. Analyt. Biochem. 181, 318–320.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2002

Authors and Affiliations

  • Catherine Copse
    • 1
  • Susan J. Fowler
    • 1
  1. 1.Amersham BiosciencesAmershamUK

Personalised recommendations