Product Pathways - DNA Damage
Phospho-Chk2 (Thr68) Antibody #2661
PhosphoSitePlus® protein, site, and accession data: Chk2
| Applications | Reactivity | Sensitivity | MW (kDa) | Source |
|---|---|---|---|---|
| W IP IF-IC F | H Mk | Endogenous | 62 | Rabbit |
Applications Key:
W=Western Blotting
IP=Immunoprecipitation
IF-IC=Immunofluorescence (Immunocytochemistry)
F=Flow Cytometry
Reactivity Key:
H=Human
Mk=Monkey
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.
Protocols
Specificity / Sensitivity
Phospho-Chk2 (Thr68) Antibody detects endogenous levels of Chk2 only when phosphorylated at threonine 68. The antibody does not cross-react with Chk2 phosphorylated at other sites.
Source / Purification
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr68 of human Chk2. Antibodies are purified by protein A and peptide affinity chromatography.
Western Blotting
Western blot analysis of extracts from Cos cells, untreated or UV-treated (100 mJ/cm2, 1 hr recovery), using Phospho-Chk2 (Thr68) Antibody
Western Blotting
Western blot analysis of extracts from HeLa cells, untreated or UV-treated (100 mJ/cm2, 1 hr recovery), using Phospho-Chk2 (Thr68) Antibody
Flow Cytometry
Flow cytometric analysis of HeLa cells, untreated (blue) and UV-treated (green), using Phospho-Chk2 (Thr68) Antibody.
IF-IC
Confocal immunofluorescent analysis of HeLa cells, untreated (left) or UV-treated (right), using Phospho-Chk2 (Thr68) Antibody (green). Actin filaments have been labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
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).
- Allen, J.B. et al. (1994) Genes Dev. 8, 2401-2415.
- Weinert, T.A. et al. (1994) Genes Dev. 8, 652-665.
- Murakami, H. and Okayama, H. (1995) Nature 374, 817-819.
- Kastan, M.B. and Lim, D.S. (2000) Nat. Rev. Mol. Cell Biol. 1, 179-186.
- Matsuoka, S. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 10389-10394.
- Melchionna, R. et al. (2000) Nat. Cell Biol. 2, 762-765.
- Ahn, J.Y. et al. (2000) Cancer Res. 60, 5934-5936.
- Lee, C.H. and Chung, J.H. (2001) J. Biol. Chem. 276, 30537-30541.
Application References
- Kohn, E. A. et al. (2002) Abrogation of the S phase DNA damage checkpoint results in S phase progression or premature mitosis depending on the concentration of 7-hydroxystaurosporine and the kinetics of Cdc25C activation. J. Biol. Chem. 277, 26553-26564. Applications: Western Blotting
- Yin, M. B. et al. (2004) Enhanced 7-ethyl-10-hydroxycamptothecin (SN-38) lethality by methylselenocysteine is associated with Chk2 phosphorylation at threonine-68 and down-regulation of cdc6 expression. Molecular Pharmacology 66, 153-160. Applications: Western Blotting
- Eastman, A. et al. (2002) A novel indolocarbazole, ICP-1, abrogates DNA damage-induced cell cycle arrest and enhances cytotoxicity: similarities and differences to the cell cycle checkpoint abrogator UCN-01. Mol. Cancer Ther. 1, 1067-1078. Applications: Western Blotting
- Lukas, C. et al. (2003) Distinct spatiotemporal dynamics of mammalian checkpoint regulators induced by DNA damage. Nat. Cell Biol. 5, 255-260. Applications: IC-IF Western Blotting
- Castedo, M. et al. (2004) The cell cycle checkpoint kinase Chk2 is a negative regulator of mitotic catastrophe. Oncogene 23, 4353-4361. Applications: Flow Cytometry IC-IF Western Blotting
- Bartucci, M. et al. (2011) Cell Death Differ , . Applications: Western Blotting
- Mallette, F.A. et al. (2012) EMBO J 31, 1865-78. Applications: IF-IC (In Cells) Western Blotting
- Niziolek-Kierecka, M. et al. (2012) Chem Res Toxicol 25, 862-72. Applications: Western Blotting
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For Research Use Only. Not For Use In Diagnostic Procedures.
