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31245
Cleaved Caspase-9 (Asp315) (D8I9E) Rabbit mAb (PE Conjugate)

Cleaved Caspase-9 (Asp315) (D8I9E) Rabbit mAb (PE Conjugate) #31245

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APPLICATIONS

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

Flow cytometric analysis of Jurkat cells, untreated (blue) or treated with Etoposide #2200 (25 μM, overnight; green), using Cleaved Caspase-9 (Asp315) (D8I9E) Rabbit mAb (PE Conjugate).

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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-Caspase-9 (Asp315) (D8I9E) Rabbit mAb (PE Conjugate) recognizes endogenous levels of caspase-9 protein only when cleaved at Asp315.

Species Reactivity:

Human

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Asp315 of human caspase-9 protein.

This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometry analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated Cleaved-Caspase-9 (Asp315) (D8I9E) Rabbit mAb #20750.

Caspase-9 (ICE-LAP6, Mch6) is an important member of the cysteine aspartic acid protease (caspase) family (1,2). Upon apoptotic stimulation, cytochrome c released from mitochondria associates with the 47 kDa procaspase-9/Apaf-1. Apaf-1 mediated activation of caspase-9 involves intrinsic proteolytic processing resulting in cleavage at Asp315 and producing a p35 subunit. Another cleavage occurs at Asp330 producing a p37 subunit that can serve to amplify the apoptotic response (3-6). Cleaved caspase-9 further processes other caspase members, including caspase-3 and caspase-7, to initiate a caspase cascade, which leads to apoptosis (7-10).

  1. Duan, H. et al. (1996) J. Biol. Chem. 271, 16720-16724.
  2. Srinivasula, S. M. et al. (1996) J. Biol. Chem. 271, 27099-27106.
  3. Liu, X. et al. (1996) Cell 86, 147-157.
  4. Li, P. et al. (1997) Cell 91, 479-489.
  5. Zou, H. et al. (1999) J. Biol. Chem. 274, 11549-11556.
  6. Srinivasula, S.M. et al. (1998) Mol Cell 1, 949-57.
  7. Deveraux, Q. L. et al. (1998) EMBO J. 17, 2215-2223.
  8. Slee, E. A. et al. (1999) J. Cell Biol. 144, 281-292.
  9. Sun, X.M. et al. (1999) J Biol Chem 274, 5053-60.
  10. MacFarlane, M. et al. (1997) J. Cell Biol. 137, 469-479.
Entrez-Gene Id
842
Swiss-Prot Acc.
P55211
For Research Use Only. Not For Use In Diagnostic Procedures.

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

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