O'Brien M A, Moravec R A, Riss T L
Promega Corporation, Madison, WI, USA.
Biotechniques. 2001 Apr;30(4):886-91. doi: 10.2144/01304pf01.
During apoptosis, the activation of a family of cysteine proteases, or caspases, results in proteolytic cleavage of numerous substrates. Antibody probes specific for neoepitopes on protein fragments generated by caspase cleavage provide a means to monitor caspase activity at the level of the individual cell. Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA repair, is a well-known substrate for caspase-3 cleavage during apoptosis. Its cleavage is considered to be a hallmark of apoptosis. Here, we demonstrate that an affinity-purified polyclonal antibody to the p85 fragment of PARP is specific for apoptotic cells. Western blots show that the antibody recognizes the 85-kDa (p85) fragment of PARP but not full-length PARP. We demonstrate a time course of PARP cleavage and DNA fragmentation in situ using the PARP p85 fragment antibody and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) in Jurkat cells treated with anti-Fas. Furthermore, our results indicate that the p85 fragment of PARP resulting from caspase cleavage during apoptosis is rapidly localized outside the condensed chromatin but not in the cytoplasm.
在细胞凋亡过程中，半胱氨酸蛋白酶家族（即胱天蛋白酶）的激活会导致众多底物的蛋白水解切割。针对胱天蛋白酶切割产生的蛋白质片段上新表位的抗体探针提供了一种在单个细胞水平监测胱天蛋白酶活性的方法。聚（ADP - 核糖）聚合酶（PARP）是一种参与DNA修复的核酶，是细胞凋亡期间胱天蛋白酶 - 3切割的著名底物。其切割被认为是细胞凋亡的一个标志。在此，我们证明一种针对PARP的p85片段亲和纯化的多克隆抗体对凋亡细胞具有特异性。蛋白质免疫印迹显示该抗体识别PARP的85 kDa（p85）片段而非全长PARP。我们使用PARP p85片段抗体和末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）在抗Fas处理的Jurkat细胞中展示了PARP切割和DNA片段化的时间进程。此外，我们的结果表明，细胞凋亡期间由胱天蛋白酶切割产生的PARP的p85片段迅速定位于浓缩染色质之外而非细胞质中。
