Detecting tyrosine-phosphorylated proteins by Western blot analysis.

The development of monoclonal antibodies (mAbs) that recognize nearly all of the phosphorylated tyrosine residues, irrespective of the surrounding sequences, enables researchers to detect the phosphorylation state of proteins through the use of anti-phosphotyrosine western blotting. The availability of this simple, reliable, nonradioactive and yet sensitive method created a boom in signal transduction research. While the methodology of how to perform an anti-phosphotyrosine western blot remains unchanged since the procedure became widely used in the early part of 1990s, steady improvements in reagents and detection technologies have allowed researchers to detect tyrosine phosphorylation quantitatively, at unprecedented sensitivity. In addition to the improvements in the western blot–based systems, powerful new phosphotyrosine detection platforms, based on proteomic technologies, are emerging rapidly. This unit will describe in detail the steps needed to perform the standard anti-phosphotyrosine western blot analysis. Curr. Protoc. Immunol. 89:11.3.1-11.3.11. © 2010 by John Wiley & Sons, Inc. Keywords: tyrosine-phosphorylated proteins; tyrosine kinase substrates; anti-phosphotyrosine western blot