Generation and use of anti-phosphotyrosine antibodies raised against bacterially expressed abl protein

Polyclonal antibodies for phosphotyrosine can be obtained by immunization with a t/abl fusion protein. These antibodies work well in immunoblotting and immunostaining experiments to identify P-Tyr proteins. For example, a 95-kDa thrombin-induced P-Tyr protein in platelets is recognized by these polyclonal antibodies but reacts poorly with other types of anti-P-Tyr antibodies (A. Golden and J.S. Brugge, 1988, personal communications). To obtain large quantities of anti-P-Tyr antibodies for preparative purposes, it is much easier to use hybridomas. An attempt to isolate hybridoma anti-P-Tyr antibodies by immunization with t/abl protein was unsuccessful, possibly because anti-P-Tyr antibodies were produced by a small percentage (approximately 2%) of the immune cells responsive to the t/abl fusion protein. Monoclonal anti-P-Tyr antibodies, however, have been produced using other types of immunogens (see Chapters [8] and [9] in this volume). The different preparations of anti-P-Tyr antibodies appear to have distinct characteristics. Test several preparations to determine the optimal reagents for each experimental need.