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Thompson and Sawtell report that the Promega Anti-PARP p85 antibody did not recognize cleaved PARP in mouse or rabbit cells in their experiments, and conclude that the results reported with this antibody by Perng et al. (1) are an artifact. The Promega antibody was generated against a peptide based on the sequence of human p85. Although the corresponding bovine sequence differs by two amino acids, the antibody reacts with both human and bovine p85 (2). The mouse and rat sequences for this region of p85 differ from the human sequence by a single amino acid that corresponds to one of the bovine amino acid differences. External testers have successfully stained mouse and rat p85 using Promega Anti-PARP p85 (2). Thus, the negative mouse results reported by Thompson and Sawtell are surprising, and call into question the validity of their negative rabbit results.
Extracts that we prepared (Fig. 1) from rabbit skin cells induced to undergo apoptosis by staurosporin (lane RS-S) contained a band of approximately 85 kD that was recognized by Anti-PARP p85, and that comigrated with the p85 band induced in human Jurkat cells by staurosporin (lane Jurkat-S) or anti-Fas antibody (lane Jurkat-F). Clearly, then, the Promega antibody recognizes the rabbit cleaved PARP p85 protein, and the argument to the contrary by Thompson and Sawtell has no merit. Their negative mouse and rabbit results apparently stemmed from technical problems, a bad batch of antibody, or some other unknown factor.