Dear Dr. Parak, To respond to your inquiry, I have used the Anti-PARP antibody with success. I stained Jurkat cells induced to undergo apoptosis with ~4 hours anti-Fas mAb treatment (I think the anti-Fas clone designation was CH-11). I pretty much followed the recommended procedure supplied with the antibody except I used a donkey anti-rabbit Cy3 conjugate from Jackson for fluorescence microscopy and I got good results. You should be able to see or print a copy of Promega's technical literature with a detailed protocol on thier web site at http://www.promega.com/tbs/tb273/tb273.pdf Regarding use for flow cytometry, I also noticed Dr. Darzynkiewcz responded (March 8) to the cytometry mailing list indicating he has used the Anti-PARP. See (Exp. Cell Res., 255, 125-132, 2000). They show immunocytochemical staining of cleaved PARP in HL-60 cells correlates with apoptotic morphology as expected. They also used the antibody for multiparameter flow and laser scanning cytometry applications. There also was a recent paper in Feb. 25th issue of Science (Perng, G-C., et al. (2000) Virus-induced neuronal apoptosis blocked by the herpes simplex virus latency-associated transcript. Science 287, 1500-1503.) showing immunohistochemical staining of rabbit neurons on trigeminal ganglia (whatever they are) using anti-PARP. A detailed procedure was not included indicating exactly how they stained rabbit tissue using a rabbit pAb as the primary antibody. I also remember seeing this antibody used for dual staining along with the TUNEL assay on a poster presented at a recent meeting and the images looked good. I remember seeing a time course comparing TUNEL and Anti-PARP on the poster indicating that PARP cleavage preceeded DNA fragmentation by TUNEL... which agrees other results reported by Li and Darzynkiewicz. It makes sense that PARP would be cleaved before extensive DNA fragmentation. I think this will be a useful marker of apoptosis. Ty Lee -----Original Message----- From: Parak, Ruqayya, Mrs <078PAR@chiron.wits.ac.za> To: cyto-inbox Date: Tuesday, March 07, 2000 12:56 AM Subject: Apoptosis quiry >Dear Ty Lee >I would like to know whether you have tried the promega antibody that >recognizes the p85 fragment of PARP. If so could you please let me >know the methodology you adhered to and the quality of your results. >Thank you. >R.Parak >Witwatersrand University >South Africa >
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