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.
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D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D005455:Fluorescent Antibody Technique; D016313:Genes, abl; D015151:Immunoblotting / Q000379:methods; D016253:Microscopy, Immunoelectron; D016374:Oncogene Proteins v-abl / Q000032:analysis* / Q000235:genetics; D010750:Phosphoproteins / Q000032:analysis*; D019000:Phosphotyrosine; D011817:Rabbits / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000032:analysis; D011994:Recombinant Proteins / Q000032:analysis; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000276:immunology
D000906:Antibodies; D016374:Oncogene Proteins v-abl; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D014443:Tyrosine