Skip to main content

Part of the book series: Neuromethods ((NM,volume 34))

  • 940 Accesses

Abstract

Neuroscience has quickly evolved to include classical neurochemical as well as molecular approaches to the study of proteins, This marriage of disciplines has been brought about not only by our need to further explore systems of specific interest, but also by the increased availability of commercial products and equipment. Protocols for the study of proteins are also numerous, and their use varies according to the experimental objectives. One of these approaches to the study of proteins, Western blot analysis —also known as electroblotting (Towbin et al., 1979) or immunoblotting (Burnette, 1981)—is a rapid and sensitive assay which combines the resolution of gel electrophoresis and the principles of immunological recognition of an antigen (e.g., the protein) by an appropriate antibody. We will begin with a discussion of the general principles and considerations of gel electrophoresis, the preparation of cell extracts, and the choice of antibodies, followed by a detailed description of the most common and reliable procedures that we have adapted and use routinely and satisfactorily. The reader wishing greater detail about alternate protocols is directed to other available sources (Harlow and Lane, 1988; Timmons and Dunbar, 1990; Ausubel et al., 1994).

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  • Ausubel, F M, Brent, M, Kingston, R E, Moore, D D, Seidman, J G, Smith, J A, and Struhl, K (eds.) (1994) Current Protocols in Molecular Biology, Wiley, New York

    Google Scholar 

  • Blo-Rad (1996) Protein Blottinng A Guide to Tranfer and Detection, Hercules, CA

    Google Scholar 

  • Blerrum, O J, Larsen, K P, and Heegaard, N H H (1988) Nonspecific binding and artifacts-specificity problems and troubleshooting with an atlas of Immunoblotting artifacts, in CRC Handbook of Immunoblotting #hotems, Vol I Technical Descriptions (Bjerrum, O J and Heegaard, N H H, eds), CRC Press, Boca Raton, FL, pp 227–254

    Google Scholar 

  • 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 radiolodinated protein A Anal Blockem 112,195–203.

    CAS  Google Scholar 

  • CLONTECH (1996) Yeast Protocols Handbook (PT3024-1) Palo Alto, CA

    Google Scholar 

  • Harlow, E and Lane, D (1988) Immunoblotting, in Antibodies A Laboratory Manual, CSH Laboratory, Cold Spring Harbor, New York, pp 471–510

    Google Scholar 

  • Kessler, S W. (1975) Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent parameters of the interaction of antibodyantigen complexes with protein A J Immunol 115, 1617–1624

    PubMed  CAS  Google Scholar 

  • Kohler, G and Milstem, C (1975) Contmuous cultures of fused cells secreting antibody of predefined specificity Nature(London) 256,495–497

    Article  PubMed  CAS  Google Scholar 

  • Kyhse-Andersen (1984) Electroblotting of multiple gels a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to introcellulose J Biochem Biophys Methods 10,203–209

    Article  PubMed  CAS  Google Scholar 

  • Laemmli, U K (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature(London) 227,680–685

    Article  PubMed  CAS  Google Scholar 

  • Penalva, M A and Salas, M. (1982) Initiation of phage phi 29 DNA replication in vitro formation of a covalent complex between the terminal protein, p3, and 5′-dAMP Proc Nat1 Acad SciUSA 79,5522–5526

    Article  CAS  Google Scholar 

  • Prmten, J. A and Sprague, G F, Jr (1994) Protein-protein interactions in the yeast pheromone response pathway. Ste5p interacts with all members of the MAP kmase cascade Genetics 138,609–619

    Google Scholar 

  • Rao, V L R, Audet, R M, and Butterworth, R F (1997) Portacaval shunting upregulates neuronal nitric oxide synthase gene and protein expression in rat cerebellum Brain Res 765,169–172

    Article  PubMed  CAS  Google Scholar 

  • Sambrook, J., Fntsch, E F, and Mamatls, T (eds.) (1989) Molecular Cloning A Laboratory Manuul CSH Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Schneppenhelm, R, Budde, IJ, Dahlmann, N, and Rautenberg, P (1991) Luminography-a new, highly sensitive visualization method for electrophoresis Electrophoresis 12, 367–372

    Article  Google Scholar 

  • Timmons, T H and Dunbar, B S. (1990) Protein blotting and immunodetection, in Methods in Enzymology, vol 282 Guide to Protein Purification (Deutscher, M P, ed), Academic Press, New York, pp 679–688

    Chapter  Google Scholar 

  • Towbin, H, Staehelm, T, and Gordon, J (1979) Electrophoretlc transfer of proteins from polyacrylamlde gels to introcellulose sheets. Procedure and some apphcatlons. Proc Nat1 Acad SCI USA 76,4350–4354

    Article  CAS  Google Scholar 

  • Wickner, W and Kornberg, A (1974) A holoenzyme form of deoxyribonucleic acid polymerase III Isolation and properties. I Biol Chem 249,6244–6249

    CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 1999 Humana Press Inc

About this protocol

Cite this protocol

Mousseau, D.D., Raghavendra Rao, V.L. (1999). Western Blot Analysis. In: Boulton, A.A., Baker, G.B., Bateson, A.N. (eds) In Vitro Neurochemical Techniques. Neuromethods, vol 34. Humana Press. https://doi.org/10.1385/0-89603-509-3:167

Download citation

  • DOI: https://doi.org/10.1385/0-89603-509-3:167

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-509-6

  • Online ISBN: 978-1-59259-639-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics