Immunoprecipitation and Western Blotting of Phosphotyrosine-Containing Proteins

  • Kathleen M. Woods Ignatoski
Part of the Methods in Molecular Biology™ book series (MIMB, volume 124)


Changes in the tyrosine phosphorylation state of a protein in response to external stimuli can have profound effects on cellular signal transduction. The addition of a phosphate group to a tyrosine residue can change a protein’ activation state or create a high affinity binding site for other proteins. Conversely, removal of a phosphate group can also change the catalytic activity of an enzyme. Tyrosine phosphorylation of cellular proteins is a rare event that can be increased growth factor addition or cellular attachment to extracellular matrix. Therefore, it is important to be able to observe changes in tyrosine phosphorylation of particular proteins under the influence of different stimuli. Tyrosine phosphorylation of proteins is difficult to detect unless external stimuli are present; even then, many proteins are phosphorylated only in response to one stimulus. Therefore, it is necessary to concentrate the protein of interest in order to observe the phosphorylation state changes between stimulated and unstimulated cells. 32P-labeling of cellular proteins can be used; however, phosphoserine and phosphothreonine are also detected along with phosphotyrosine. Phosphoamino acid analysis can be helpful, but it is not quantitative because acid hydrolysis, which breaks down the proteins into individual amino acids, can remove the phosphate group from the tyrosine. Therefore, other methods of detecting changes in tyrosine phosphorylation states have been developed.


Tyrosine Phosphorylation Ammonium Persulfate Sepharose Bead Tyrosine Phosphorylated Protein High Affinity Binding Site 
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  1. 1.
    Ross A. H., Baltimore D., and Eisen H. N. (1981) Phosphotyrosine-containing proteins isolated by affinity chromatography with antibodies to synthetic hapten. Nature (London) 294, 654–656.CrossRefGoogle Scholar
  2. 2.
    Frackleton A. R., Ross A. H., and Eisen H. N. (1983) Characterization and use of monoclonal antibodies for isolation of phosphotyrosyl proteins from retrovirus-transformed cells and growth factor-stimulated cells. Mol. Cell. Biol. 3, 1343–1352.Google Scholar
  3. 3.
    Wang J. Y. J. (1991) Generation and use of anti-phosphotyrosine antibodies raised against bacterially expressed abl protein. Meth. Enzymol. 201, 53–65.PubMedCrossRefGoogle Scholar
  4. 4.
    Frackleton A. R., Psner M., Kannan B., and Mermelstein F. (1991) Generation of monoclonal antibodies and their use for affinity purification of phosphotyrosine-containing proteins. Meth. Enzymol. 201, 79–92.CrossRefGoogle Scholar
  5. 5.
    White M. F. and Backer J. M. (1991) Preparation and use of anti-phosphotyrosine antibodies to study structure and function of insulin receptor. Meth. Enzymol. 201, 65–79.PubMedCrossRefGoogle Scholar
  6. 6.
    Wang J. Y. J. (1988) Antibodies for phosphotyrosine: analytical and preparative tool for tyrosyl-phosphorylated proteins. Anal. Biochem. 172, 1–7.PubMedCrossRefGoogle Scholar
  7. 7.
    Woods Ignatoski K. M. and Verderame M. F. (1996) Lysis buffer composition dramatically affects extraction of phosphotyrosine-containing proteins, BioTechniques 20, 794–796.Google Scholar
  8. 8.
    Helenius A., McCaslin D., R., Fries E., and Tanford C. (1979) Properties of detergents. Meth. Enzymol. 56, 734–749.PubMedCrossRefGoogle Scholar
  9. 9.
    Hjelmeland L. M. and Chrambrach A. (1984) Solubilization of functional membrane-bound receptors, in Membranes, Detergents, and Receptor Solubilization, Alan R. Liss, New York, pp. 35–40.Google Scholar
  10. 10.
    Roda A., Hofmann A. F., and Mysels K. J. (1983) The influence of bile salt structure on self association in aqueous solutions. J. Biol. Chem. 258, 6362–6370.PubMedGoogle Scholar
  11. 11.
    Rudzki J. E. and Peters K. S. (1984) Picosecond absorption studies on rhodopsin and isorhodopsin in detergent and native membranes. Biochemistry 23, 3843–3848.PubMedCrossRefGoogle Scholar
  12. 12.
    Wisdom G. B. (1994) Protein blotting, in Basic Protein andPeptide Protocols, 1st ed. (Walker J. M., ed.), Methods in Molecular Biology, vol. 32, Humana, Totowa, NJ, pp. 207–213.Google Scholar
  13. 13.
    Page M. and Thorpe R. (1996) Protein blotting by electroblotting, in The Protein Protocols Handbook, 1st ed. (Walker J. M., ed.), Humana, Totowa, NJ, pp. 245–258.CrossRefGoogle Scholar
  14. 14.
    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
  15. 15.
    Promega Protein Guide: Tips and Techniques, pp. 13–22 (1993), Promega, Madison, WI.Google Scholar
  16. 16.
    Protein Blotting Protocols for Immobilon-P Transfer Membrane, pp. 1–7 (1991), Millipore, Bedford, MA.Google Scholar
  17. 17.
    Protein Blotting: A Guide to Transfer and Detection, pp. 6–49 (1991), Bio-Rad, Richmond, CA.Google Scholar
  18. 18.
    ProtoBlot Western Blot AP System, Technical Manual (1987), pp. 1–15, Promega, Madison, WI.Google Scholar
  19. 19.
    Woods K. M. and Verderame M. F. (1994) Autophosphorylation is required for kinase activity and transformation ability of proteins encoded by host-range alleles of v-src. J. Virol. 68, 7267–7274.PubMedGoogle Scholar

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© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • Kathleen M. Woods Ignatoski

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