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Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb (PE Conjugate) #12812

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

Flow cytometric analysis of HeLa cells, untreated (blue) or UV-treated (green), using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb (PE Conjugate).

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Protocol

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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
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Product Usage Information

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 antibodies. Protect from light. Do not freeze.
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Specificity / Sensitivity

Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb (PE Conjugate) detects endogenous levels of Chk2 only when phosphorylated at Thr68.


Species Reactivity: Human
Species predicted to react based on 100% sequence homology: Monkey
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Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr68 of human Chk2.

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Product Description

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 Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb #2197.


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Background

Chk2 is the mammalian orthologue of the budding yeast Rad53 and fission yeast Cds1 checkpoint kinases (1-3). The amino-terminal domain of Chk2 contains a series of seven serine or threonine residues (Ser19, Thr26, Ser28, Ser33, Ser35, Ser50, and Thr68) each followed by glutamine (SQ or TQ motif). These are known to be preferred sites for phosphorylation by ATM/ATR kinases (4,5). After DNA damage by ionizing radiation (IR), UV irradiation, or hydroxyurea treatment, Thr68 and other sites in this region become phosphorylated by ATM/ATR (5-7). The SQ/TQ cluster domain, therefore, seems to have a regulatory function. Phosphorylation at Thr68 is a prerequisite for the subsequent activation step, which is attributable to autophosphorylation of Chk2 at residues Thr383 and Thr387 in the activation loop of the kinase domain (8).


1.  Allen, J.B. et al. (1994) Genes Dev. 8, 2401-2415.

2.  Weinert, T.A. et al. (1994) Genes Dev. 8, 652-665.

3.  Murakami, H. and Okayama, H. (1995) Nature 374, 817-819.

4.  Kastan, M.B. and Lim, D.S. (2000) Nat. Rev. Mol. Cell Biol. 1, 179-186.

5.  Matsuoka, S. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 10389-10394.

6.  Melchionna, R. et al. (2000) Nat. Cell Biol. 2, 762-765.

7.  Ahn, J.Y. et al. (2000) Cancer Res. 60, 5934-5936.

8.  Lee, C.H. and Chung, J.H. (2001) J. Biol. Chem. 276, 30537-30541.


Entrez-Gene Id 11200
Swiss-Prot Acc. O96017
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Data Sheets & Documentation

For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 5,675,063.

12812
Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb (PE Conjugate)
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