Detecting and Quantifying pADPr In Vivo

Abstract

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 depletion of NAD⁺ and mitochondrial dysfunction, potentially leading to cell death. Since PARP overactivation and increases in free pADPr have been identified as key contributors 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.

Keywords: ADP-ribose polymerase; Poly(ADP-ribose) polymerase; Poly(ADP-ribose) polymers; Poly(ADP-ribose) synthetase; Poly-ADP-ribosylation.