PE Mouse Anti-Cleaved PARP (Asp214)
Clone F21-852 (RUO)
- Brand BD Pharmingen™
- Vol. Per Test 20 µl
- Isotype Mouse IgG1, κ
- Reactivity Human (QC Testing)
Intracellular staining (flow cytometry) (Routinely Tested)
- Immunogen Human cleaved PARP
- Storage Buffer Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
- Regulatory Status RUO
Regulatory Status Legend
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.
- Format PE
- Excitation Source Blue 488 nm,Green 532 nm,Yellow/Green 561 nm
- Excitation Max 496 nm
- Emission Max 578 nm
R-phycoerythrin (PE) is an accessory photosynthetic pigment found in red algae. It exists in vitro as a 240-kDa protein with 23 phycoerythrobilin chromophores per molecule. This makes PE one of the brightest fluorochromes for flow cytometry applications, but its photobleaching properties make it unsuitable for fluorescence microscopy.
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Preparation and Storage
The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.
The antibody was conjugated with R-PE under optimum conditions, and unconjugated antibody and free PE were removed.
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
- This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
- Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- 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.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
Camptothecin (an extract of the Chinese tree Camptotheca acuminata) is a potent inhibitor of topoisomerase I, a molecule required for DNA synthesis. Camptothecin has been shown to induce apoptosis in a dose dependent manner in vitro. Camptothecin is used at BD Biosciences Pharmingen as a general method for inducing apoptosis.
-1.0 µM stock solution of camptothecin (Sigma; Cat. No. C-9911) in DMSO.
-Jurkat cell line (ATCC TIB-152), proliferating, at 1 x 10^6 cells/ml.
-Either Cytofix/Cytoperm™ Fixation/Permeablization Kit (Cat. No. 554714) or Cytofix/Cytoperm™ solution (Cat. No. 554722) plus Perm/Wash™ buffer (Cat. No. 554723).
1. Add camptothecin (4-6 mM final concentration) per 1 x 10^6 proliferating Jurkat cells. If desired, a control aliquot of untreated cells should also be prepared.
2. Incubate the cells for 4 hours at 37°C.
3. Wash the cells (camptothecin-treated and control aliquots) twice with cold PBS; then resuspend them in Cytofix/Cytoperm™ solution at 2 x 10^6 cells/ml.
4. Incubate the cells for 20 minutes on ice.
5. Pellet the cells, and aspirate and discard the Cytofix/Cytoperm™ solution.
6. Wash the cells twice at room temperature with 0.5 ml Perm/Wash™ buffer per 1 x 10^6 cells, and discard the supernatants.
7. Resuspend the cells in Perm/Wash™ buffer at 10 x 10^6 /ml.
8. Aliquot test samples of 1 x 10^6 cells per 100-µl test.
9. Add 20 µl antibody per test, and incubate for 30 minutes at room temperature.
10. Wash each test in 1.0 ml Perm/Wash™ Buffer and discard the supernatant.
11. Resuspend each test in 0.5 ml Perm/Wash™ Buffer and analyze by flow cytometry.