Poly (ADP-ribose) polymerase cleavage monitored in situ in apoptotic cells

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.

MeSH terms

  • Antibodies
  • Antibody Specificity
  • Apoptosis*
  • Blotting, Western
  • Cytoplasm / chemistry
  • DNA Fragmentation
  • Fluorescent Dyes
  • Humans
  • In Situ Nick-End Labeling*
  • Indoles
  • Jurkat Cells
  • Poly(ADP-ribose) Polymerases / immunology
  • Poly(ADP-ribose) Polymerases / metabolism*
  • fas Receptor / analysis
  • fas Receptor / immunology


  • Antibodies
  • Fluorescent Dyes
  • Indoles
  • fas Receptor
  • DAPI
  • Poly(ADP-ribose) Polymerases