Wang J Y
Methods Enzymol. 1991;201:53-65. doi: 10.1016/0076-6879(91)01008-p.
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.
用于磷酸酪氨酸的多克隆抗体可通过用t/abl融合蛋白免疫获得。这些抗体在免疫印迹和免疫染色实验中能很好地识别磷酸化酪氨酸蛋白。例如，这些多克隆抗体可识别血小板中一种95 kDa的凝血酶诱导的磷酸化酪氨酸蛋白，但与其他类型的抗磷酸化酪氨酸抗体反应较差（A. 戈尔登和J.S. 布鲁格，1988年，个人交流）。为了获得大量用于制备目的的抗磷酸化酪氨酸抗体，使用杂交瘤要容易得多。通过用t/abl蛋白免疫来分离杂交瘤抗磷酸化酪氨酸抗体的尝试未成功，可能是因为抗磷酸化酪氨酸抗体是由一小部分（约2%）对t/abl融合蛋白有反应的免疫细胞产生的。然而，已经使用其他类型的免疫原制备出了单克隆抗磷酸化酪氨酸抗体（见本卷第[8]章和第[9]章）。不同的抗磷酸化酪氨酸抗体制剂似乎具有不同的特性。测试几种制剂，以确定满足每种实验需求的最佳试剂。
