Product Pathways - DNA Damage

Phospho-Chk1/2 Antibody Sampler Kit #9931

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Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  Mi=Mink  Pg=Pig

Specificity / Sensitivity

Each phospho-specific Chk1 antibody detects endogenous levels of Chk1 when phosphorylated at the indicated site (Ser317 or Ser345), and each phospho-specific Chk2 antibody detects endogenous levels Chk2 only when phosphorylated at the indicated site (Ser19, Ser33/35, Thr68, Thr387 or Thr432). Chk1 Antibody #2345 detects endogenous levels of total Chk1. Chk2 Antibody #2662 detects endogenous levels of total Chk2.

Western Blotting

Western blot analysis of extracts from UV or MMS treated C6 cells, and HU treated HeLa cells, using Phospho-Chk1 (Ser345) Antibody #2341.

Western Blotting

Western blot analysis of extracts from UV or HU treated MvILu cells, using Phospho-Chk1 (Ser317) Antibody #2344.

Western Blotting

Western blot analysis of extracts from 293, MvILu, C6, HeLa and COS cells, using Chk1 Antibody #2345.

Western Blotting

Western blot analysis of COS cell extracts untreated and treated with UV; or transfected with Wild-type Chk2, Chk2 (T68A), Chk2 (T26S28A) and Chk2 (S33S35A), using Phospho-Chk2 (Thr68) Antibody #2661.

Western Blotting

Western blot analysis of extracts from control, UV treated and doxorubicin (0.5 µM) treated 293 cells, using Phospho-Chk2 (Thr68) Antibody #2661.

Western Blotting

Western blot analysis of COS cell extracts, control (lane 1), transfected with Wild-type Chk2 (lane 2), Chk2 (S19A) (lane 3), Chk2 (T26S28A) (lane 4), Chk2 (S33S35A) (lane 5) and Chk2 (T68A) (lane 6), using Phospho-Chk2 (Ser33/35) Antibody #2665.

Western Blotting

Western blot analysis of extracts from UV treated HeLa cells, using Phospho-Chk2 (Ser33/35) Antibody #2665.

Western Blotting

Western blot analysis of COS cell extracts, control (lane 1), transfected with Wild-type Chk2 (lane 2), Chk2 (S19A) (lane 3), Chk2 (T26S28A) (lane 4), Chk2 (S33S35A) (lane 5) and Chk2 (T68A) (lane 6), using Phospho-Chk2 (Ser19) Antibody #2666.

Western Blotting

Western blot analysis of extracts from UV treated Hela cells, using Phospho-Chk2 (Ser19) Antibody #2666.

Western Blotting

Western blot analysis of extracts from untreated or UV treated HeLa cells, using Phospho-Chk2 (Thr432) Antibody #2667.

Western Blotting

Western blot analysis of extracts from untreated or UV treated 293 and COS cells, using Phospho-Chk2 (Thr387) Antibody #2668.

IHC-P (paraffin)

Immunohistochemical staining of phosphorylated Chk1 in paraffin-embedded human breast carcinoma showing nuclear localization, using Phospho-Chk1 (Ser317) Antibody #2344.

IHC-P (paraffin)

Immunohistochemical staining of phosphorylated Chk2 in paraffin-embedded human breast carcinoma showing nuclear localization, using Phospho-Chk2 (Thr68) Antibody #2661.

IHC-P (paraffin)

Immunohistochemical staining of phosphorylated Chk2 in paraffin-embedded human breast carcinoma showing nuclear localization, using Phospho-Chk2 (Ser19) Antibody #2666.

IC-ABC

Immunofluorescent detection of phosphorylated Chk1 in untreated (left) and UV treated (right) HeLa cells, using Phospho-Chk1 (Ser317) Antibody #2344.

IF-IC

Immunofluorescent detection of phospho-Chk2 (Thr68) in untreated (left) and UV treated (right) HeLa cells, using Phospho-Chk2 (Thr68) Antibody #2661.

IF-IC

Immunofluorescent detection of phospho-Chk2 in untreated (A), UV treated (B) and doxorubicin treated (C) Hela cells, using Phospho-Chk2 (Ser33/35) Antibody #2665.

Source / Purification

Polyclonal antibodies are produced by immunizing rabbits with synthetic phospho-peptides (KLH coupled) corresponding to residues surrounding Ser317 or Ser345 of human Chk1 and Ser19, Ser33/35, Thr68, Thr387 or Thr432 of human Chk2. Chk1 Antibody #2345 is produced by immunizing rabbits with a synthetic peptide (KLH coupled) corresponding to residues around Ser296 of human Chk1. Chk 2 Antibody #2662 is produced by immunizing rabbits with a synthetic peptide (KLH coupled) corresponding to amino-terminal residues of human Chk2. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.

Background

Chk1 kinase acts downstream of ATM/ATR kinase to play an important role in DNA damage checkpoint control, embryonic development and tumor suppression (1). Activation of Chk1 involves phosphorylation of Ser317 and Ser345 and occurs in response to blocked DNA replication and certain forms of genotoxic stress (2). Chk1 is also phosphorylated at Ser280 and Ser296 following DNA damage. Activated Chk1 can inactivate cdc25C via phosphorylation at Ser216, blocking the activation of cdc2 and transition into mitosis (3). Chk1 can also phosphorylate p53 at Ser20 in vitro (4).

Chk2 is the mammalian homologue of the budding yeast Rad53 and fission yeast Cds1 checkpoint kinases (5-7). The amino-terminal domain of Chk2 contains a series of seven serine or threonine residues (Ser19, Thr26, Ser28, Ser33, Ser35, Ser50 and Thr68) followed by glutamine (SQ or TQ motif). These are known to be preferred sites for phosphorylation by ATM/ATR kinases (8). Indeed, after DNA damage by ionizing radiation (IR), UV irradiation and DNA replication blocked by hydroxyurea, Thr68 and other sites in this region become phosphorylated by ATM/ATR (9-11). 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 on residues Thr383 and Thr387 in the activation loop of the kinase domain (12).

1. Martinho, R.G. et al. (1998) EMBO J. 17, 7239-7249.
2. Zhao, H. et al. (2001) Mol. Cell. Biol. 21, 4129-4139.
3. Zeng, Y. et al. (1998) Nature 395, 507-510.
4. Shieh, S. et al. (2000) Genes Dev. 14, 289-300.
5. Allen, J. B. et al. (1994) Genes Dev. 8, 2401-2415.
6. Weinert, T. A. et al. (1994) Genes Dev. 8, 652-665.
7. Murakami, H. and Okayama, H. (1995) Nature 374, 817-819.
8. Kastan, M.B. and Lim, D.S. (2000) Nat. Rev. Mol. Cell Biol. 1, 179-186.
9. Matsuoka, S. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 10389-10394.
10. Melchionna, R. et al. (2000) Nat. Cell Biol. 2, 762-765.
11. Ahn, J. Y. et al. (2000) Cancer Res. 60, 5934-5936.

Application References

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