Isolation of tyrosine-phosphorylated proteins and generation of monoclonal antibodies.

The methods used above allow one to generate the tools to study individual phosphotyrosine-containing proteins. While all of the substrates we have identified in this way contain phosphotyrosine, this cannot be assumed. Some polypeptides may bind to the anti-phosphotyrosine column because of association with other phosphotyrosine-containing proteins or by nonspecific binding to the anti-phosphotyrosine affinity column. The antibodies generated to these substrates allow one to assess potential questions directly. At a minimum, phosphoamino acid analysis must be performed on the candidate substrate labeled in intact cells with 32PO4 and precipitated with the antibody to the substrate. Methods for this can be found in Cooper and Hunter.