Lai Yi-Chen, Aneja Rajesh K, Satchell Margaret A, Clark Robert S B
Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA.
Methods Mol Biol. 2011;780:117-34. doi: 10.1007/978-1-61779-270-0_8.
Poly(ADP-ribose) polymerases (PARP) participate in diverse biological processes contributing to cellular homeostasis or exacerbating injury. PARP catalyzes the addition of ADP-ribose molecules (pADPr) to the target proteins, a process termed poly-ADP-ribosylation. Overactivation of PARP, as reflected by increased poly-ADP-ribosylation, accumulation of pADPr-modified proteins or free pADPr, contributes to the depletion of NAD(+) and mitochondrial dysfunction, potentially leading to cell death via apoptosis or necrosis. Since PARP over-activation has been identified as a key contributor to the pathobiology of many diseases, monitoring PARP 1 activation by detecting and quantifying pADPr may provide valuable mechanistic insights as well as facilitating therapeutic drug monitoring for PARP inhibitors.Several non-isotopic immunodetection methods for quantifying pADPr are discussed: western blotting of poly-ADP-ribosylated proteins, cellular localization of pADPr by immunohistochemistry, quantification of pADPr by enzyme-linked immunoassay and small-scale two-dimensional gel electrophoresis.
聚（ADP-核糖）聚合酶（PARP）参与多种生物学过程，对细胞稳态或加重损伤有影响。PARP催化将ADP-核糖分子（pADPr）添加到靶蛋白上，这一过程称为多聚ADP-核糖基化。PARP的过度激活，表现为多聚ADP-核糖基化增加、pADPr修饰蛋白或游离pADPr的积累，会导致NAD(+)耗竭和线粒体功能障碍，可能通过凋亡或坏死导致细胞死亡。由于PARP过度激活已被确定为许多疾病病理生物学的关键因素，通过检测和定量pADPr来监测PARP 1激活可能提供有价值的机制见解，并有助于对PARP抑制剂进行治疗药物监测。本文讨论了几种用于定量pADPr的非同位素免疫检测方法：多聚ADP-核糖基化蛋白的蛋白质印迹法、通过免疫组织化学对pADPr进行细胞定位、通过酶联免疫测定法定量pADPr以及小规模二维凝胶电泳法。
