Fran, There are other anti-caspase-3 antibodies out there. 1) Merck-Frost in Canada (Don Nicholson's group) has reported on an antibody directed against the active form of caspase-3. It was raised against crystal grade recombinant caspase-3 as the antigen. To my knowledge, this one is not commercially available. 2) IDUN Pharmaceuticals (Kevin Tomaselli's group) has reported on an anti-caspase-3 directed against a peptide antigen spanning from the center of the active site cys to the C-terminus of the caspase-cleavage site (i.e. where caspase-3 is cleaved to form the ~p17 and ~p12 subunits). I do not know if this specific preparation of affinity purified rabbit pAb is commercially available; however, R&D Systems offers one using the same sequence of peptide antigen. 3) Pharmingen's antibody (if I remember correctly) uses a peptide antigen that is slightly different than the sequence used by IDUN. I have seen reports that there are problems with background using this antibody as well as limitations for IHC applications in formalin fixed paraffin embedded sections. Alternatives might be to use a cell permeable fluorescent caspase-3 substrate (such as the PhiPhiLux substrate sold by OncoImmunin see phiphilux.com) if you are looking at living cells or another marker antibody that indicates caspase-3 cleavage has occured (such as Anti-PARP p85 Fragment pAb sold by Promega see http://www.promega.com). Promega has examples of immunocytochemical staining of Anti-Fas treated Jurkat cells; but, no cytometry data are posted. Good Luck, Ty Lee tylee@itis.com -----Original Message----- From: frances gibson <fgibson@sghms.ac.uk> To: cyto-inbox Date: Friday, August 20, 1999 4:37 PM Subject: caspase-3 > >Dear all > >We have been using a anti-Caspase-3 rabbit polyclonal ab conjugated >with PE (this is supposed to bind to the active form only) by flow. >We've looked for caspase-3 activation in camptothecin or etoposide >treated Jurkatt cells (cytospins confirm apoptosis in these >cells) after fixation and perm, without success. This ab is from >Pharmingen. > >Does anyone have experience with looking for caspase-3 activation by >flow ? Are there other antibodies available ? > >Any comments would be much appreciated. > >Fran Gibson >Dept Haematology >St. George's Hospital Medical School >London > > >
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