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67495
Cleaved PARP (Asp214) (D6X6X) Rabbit mAb (Rodent Specific) (PE Conjugate)

Cleaved PARP (Asp214) (D6X6X) Rabbit mAb (Rodent Specific) (PE Conjugate) #67495

APPLICATIONS

REACTIVITY SENSITIVITY MW (kDa) Isotype
M R Endogenous Rabbit IgG
Flow Cytometry

Flow cytometric analysis of serum-starved Neuro-2a cells, untreated (blue) or treated with Staurosporine #9953 (1 μM, 3 hr; green) using Cleaved PARP (Asp214) (D6X6X) Rabbit mAb (Rodent Specific) (PE Conjugate) (solid lines) or concentration matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed lines).

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Flow Cytometry, Methanol Permeabilization Protocol for Direct Conjugates

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 16% Formaldehyde (methanol free).
  3. 100% methanol.
  4. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Fixation

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells in 0.5-1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
  3. Fix for 15 min at room temperature.
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS.

C. Permeabilization

  1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
  2. Incubate 30 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

  1. Aliquot desired number of cells into tubes or wells.
  2. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
  3. Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
  6. Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).

E. Optional DNA Dye

  1. Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
  2. Incubate for at least 5 min at room temperature.
  3. Analyze cells in DNA staining solution on flow cytometer.

posted July 2009

revised June 2017

Protocol Id: 407

Application Dilutions
Flow Cytometry 1:50
Storage:

Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.

Cleaved PARP (Asp214) (D6X6X) Rabbit mAb (Rodent Specific) (PE Conjugate) recognizes endogenous levels of the large fragment (89 kDa) of rodent PARP protein only when cleaved at Asp214.

Species Reactivity:

Mouse, Rat

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Asp214 of rodent PARP1 protein.

This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometric analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated Cleaved PARP (Asp214) (D6X6X) Rabbit mAb (Rodent Specific)) #94885.

PARP, a 116 kDa nuclear poly (ADP-ribose) polymerase, appears to be involved in DNA repair in response to environmental stress (1). This protein can be cleaved by many ICE-like caspases in vitro (2,3) and is one of the main cleavage targets of caspase-3 in vivo (4,5). In human PARP, the cleavage occurs between Asp214 and Gly215, which separates the PARP amino-terminal DNA binding domain (24 kDa) from the carboxy-terminal catalytic domain (89 kDa) (2,4). PARP helps cells to maintain their viability; cleavage of PARP facilitates cellular disassembly and serves as a marker of cells undergoing apoptosis (6).

  1. Satoh, M.S. and Lindahl, T. (1992) Nature 356, 356-358.
  2. Lazebnik, Y. A. et al. (1994) Nature 371, 346-347.
  3. Cohen, G.M. (1997) Biochem. J. 326, 1-16.
  4. Nicholson, D. W. et al. (1995) Nature 376, 37-43.
  5. Tewari, M. et al. (1995) Cell 81, 801-809.
  6. Oliver, F.J. et al. (1998) J. Biol. Chem. 273, 33533-33539.
Entrez-Gene Id
11545
Swiss-Prot Acc.
P11103
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.

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Product # Size Price
67495S
100 µl (50 tests) $ 320.0