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Immunoprecipitation and Western Blotting of Phosphotyrosine-Containing Proteins

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

Abstract

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.

Keywords

Tyrosine Phosphorylation Ammonium Persulfate Sepharose Bead Tyrosine Phosphorylated Protein High Affinity Binding Site 
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.

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Copyright information

© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • Kathleen M. Woods Ignatoski

There are no affiliations available

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