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FITC Mouse anti-Cleaved PARP (Asp214)
FITC Mouse anti-Cleaved PARP (Asp214)
Analysis of cleaved PARP in activated human T leukemia cells.  Jurkat cells were either treated with Camptothecin (right panel) or untreated (left panel).  The cells were fixed and permeabilized, then stained with FITC Mouse anti-Cleaved PARP (Cat. No. 558576) according to the protocol included in this data sheet.  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.  
Analysis of cleaved PARP in activated human T leukemia cells.  Jurkat cells were either treated with Camptothecin (right panel) or untreated (left panel).  The cells were fixed and permeabilized, then stained with FITC Mouse anti-Cleaved PARP (Cat. No. 558576) according to the protocol included in this data sheet.  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.  
Product Details
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BD Pharmingen™
Asp214
Human (QC Testing), Mouse (Tested in Development)
Mouse IgG1, κ
Human cleaved PARP
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
142, 11545
AB_647179
Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with FITC under optimum conditions, and unreacted FITC was removed.

Recommended Assay Procedures

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.

Materials

        1.0 mM 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 BD Cytofix/Cytoperm™ Fixation/Permeablization Kit (Cat. No. 554714) or BD Cytofix/Cytoperm™ solution (Cat. No. 554722) plus BD Perm/Wash™ buffer (Cat. No. 554723).

Procedure

        1.        Add Camptothecin (4-6 μM 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 4-6 hours at 37°C.

        3.        Wash the cells (Camptothecin-treated and control aliquots) twice with cold PBS; then resuspend them in BD 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 BD Cytofix/Cytoperm™ solution.

        6.        Wash the cells twice at room temperature with 0.5 ml BD Perm/Wash™ buffer per 1 x 10^6 cells, centrifuge and discard the supernatants.

        7.        Resuspend the cells in BD 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, protected from light.

      10.        Wash each test in 1.0 ml BD Perm/Wash™ Buffer, centrifuge and discard the supernatant.

      11.        Resuspend each test in 0.5 ml BD Perm/Wash™ Buffer and analyze by flow cytometry.

Product Notices

  1. 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).
  2. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  3. 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.
  4. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  5. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
558576 Rev. 2
Antibody Details
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F21-852

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.

558576 Rev. 2
Format Details
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FITC
Fluorescein (FITC) is part of the BD blue family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 494-nm and an emission maximum (Em Max) at 518-nm. FITC is designed to be excited by the Blue laser (488-nm) and detected using an optical filter centered near 520 nm (e.g., a 530/30-nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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FITC
Blue 488 nm
494 nm
518 nm
558576 Rev.2
Citations & References
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Development References (5)

  1. Amé J-C, Spenlehauer C, de Murcia G. The PARP superfamily. Bioessays. 2004; 26:882-893. (Biology).
  2. Boulares AH, Yakovlev AG, Ivanova V, et al. Role of Poly(ADP-ribose) polymerase (PARP) cleavage in apoptosis. Caspase 3-resistant PARP mutant increases rates of apoptosis in transfected cells. J Biol Chem. 1999; 274(33):22932-22940. (Biology).
  3. Cherney BW, McBride OW, Chen D, et al. cDNA sequence, protein structure, and chromosomal location of the human gene for poly(ADP-ribose) polymerase. Proc Natl Acad Sci U S A. 1987; 84(23):8370-8374. (Biology). View Reference
  4. 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).
  5. Soldani G, Scovassi AI. Poly(ADP-ribose) polymerase-1 cleavage during apoptosis: an update. Apoptosis. 2002; 7:321-328. (Biology).
View All (5) View Less
558576 Rev. 2

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