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Western blot analysis of PARP (cleavage site-specific). Jurkat cells were either left untreated (lanes 1-3) or treated with camptothecin (4 µM, 4 hours) to induce apoptosis (lanes 4-6). Lysates were probed with anti-PARP (clone F21-852, Cat. No. 552597) at concentrations of 0.25 (lanes 1, 4), 0.125 (lanes 2, 5), and 0.06 µg/ml (lanes 3, 6). Cleaved PARP is identified as a band of ~89 kDa in only the treated cells.
BD Pharmingen™ Purified Mouse Anti-Cleaved PARP (Asp214)
Regulatory Status Legend
Any use of products other than the permitted use without the express written authorization of Becton, Dickinson and Company is strictly prohibited.
Description
PARP (Poly [ADP-Ribose] Polymerase) is a 113-kDa nuclear chromatin-associated enzyme that catalyzes the transfer of ADP-ribose units from NAD+ to a variety of nuclear proteins including topoisomerases, histones, and PARP itself. The catalytic activity of PARP is increased in cells following DNA damage, and PARP is thought to play an important role in mediating the normal cellular response to DNA damage. Additionally, PARP is a target of the caspase protease activity associated with apoptosis. The PARP protein consists of an N-terminal DNA-binding domain (DBD) and a C-terminal catalytic domain separated by a central automodification domain. During apoptosis, Caspase-3 cleaves PARP at a recognition site (Asp Glu Val Asp Gly) in the DBD to form 24- and 89-kDa fragments. This process separates the DBD (which is mostly in the 24-kDa fragment) from the catalytic domain (in the 89-kDa fragment) of the enzyme, resulting in the loss of normal PARP function. It has been proposed that inactivation of PARP directs DNA-damaged cells to undergo apoptosis rather than necrotic degradation, and the presence of the 89-kDa PARP cleavage fraction is considered to be a marker of apoptosis.
A peptide corresponding to the N-terminus of the cleavage site (Asp 214) of human PARP was used as the immunogen. The F21-852 monoclonal antibody reacts only with the 89-kDa fragment of human PARP-1 that is downstream of the Caspase-3 cleavage site (Asp214) and contains the automodification and catalytic domains. It does not react with intact human PARP-1. Cross-reactivity with other members of the PARP superfamily is unknown. Recognition of cleaved PARP in mouse cells has been demonstrated, and it may also cross-react with a number of other species due to the conserved nature of the molecule.
Preparation And Storage
Store the antibody, component No. 51-9000017, at 4°C. Store the lysate, component N,o. 51-16606N, at -20°C.
Recommended Assay Procedures
Camptothecin treated Jurkat lysate [50 µg (1 µg/µl)] is provided as a positive control (51-16606N; store lysate at -20°C). Additional Jurkat lysate is available untreated (Cat. No. 611451) or as a set containing both untreated and campotothecin treated lysates (Cat. No. 550959) as ready-to-use western blot controls. Additional applications which are not not routinely tested at BD Biosciences Pharmingen include immunoprecipitation (2 µg/300 µg of lysates) and flow cytometry. The directly conjugated formats of the clone are recommended for flow cytometry. (Cat. No. 552933, and please inquire.)
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
Description | Quantity/Size | Part Number | EntrezGene ID |
---|---|---|---|
Purified Mouse Anti-Cleaved PARP | 50 µg (3 ea) | 51-9000017 | N/A |
Camptothecin Treated Jurkat Lysate | 50 µg (1 ea) | 51-16606N | N/A |
Development References (6)
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D'Amours D, Desnoyers S, D'Silva I, Poirier GG. Poly(ADP-ribosyl)ation reactions in the regulation of nucelar functions. Biochem J. 1999; 342:249-268. (Biology).
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Kaufmann SH, Desnoyers S, Ottaviano Y, Davidson NE, Poirier GG. Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis. Cancer Res. 1993; 53(17):3976-3985. (Biology). View Reference
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Lamarre D, Talbot B, Leduc Y, Muller S, Poirier G. Production and characterization of monoclonal antibodies specific for the functional domains of poly(ADP-ribose) polymerase. Biochem Cell Biol. 1986; 64(4):368-376. (Biology). View Reference
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Lamarre D, Talbot B, de Murcia G, et al. Structural and functional analysis of poly(ADP ribose) polymerase: an immunological study. Biochim Biophys Acta. 1988; 950(2):147-160. (Biology). View Reference
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Patel T, Gores GJ, Kaufmann SH. The role of proteases during apoptosis. FASEB J. 1996; 10(5):587-597. (Biology). View Reference
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Tewari M, Quan LT, O'Rourke K, et al. Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell. 1995; 81(5):801-809. (Biology). View Reference
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Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.