Cell Signaling Technology

Product Pathways - DNA Damage

Phospho-Chk1/2 Antibody Sampler Kit #9931

Kit Includes Quantity Applications Reactivity MW (kDa) Isotype
Phospho-Chk1 (Ser317) Antibody #2344 40 µl W H R Mk Mi 56 Rabbit
Phospho-Chk1 (Ser345) (133D3) Rabbit mAb #2348 40 µl W IF-IC F H M R Mk 56 Rabbit IgG
Phospho-Chk1 (Ser296) Antibody #2349 40 µl W H Mk 56 Rabbit
Chk1 (2G1D5) Mouse mAb #2360 40 µl W H M R Mk 56 Mouse IgG1
Phospho-Chk2 (Ser19) Antibody #2666 40 µl W IHC-P H 62 Rabbit
Phospho-Chk2 (Ser33/35) Antibody #2665 40 µl W H Mk 62 Rabbit
Phospho-Chk2 (Thr68) Antibody #2661 40 µl W IP IF-IC F H Mk 62 Rabbit
Chk2 Antibody #2662 40 µl W IP H M R Mk 62 Rabbit
Phospho-Chk2 (Ser516) Antibody #2669 40 µl W H 62 Rabbit
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl Goat

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
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Specificity / Sensitivity

Each phospho-specific Chk1 antibody detects endogenous levels of Chk1 when phosphorylated at the indicated site (Ser296, Ser317 or Ser345), and each phospho-specific Chk2 antibody detects endogenous levels Chk2 only when phosphorylated at the indicated site (Ser19, Ser33/35, Thr68, and Ser516). Chk1 (2G1D5) Mouse mAb #2360 detects endogenous levels of total Chk1. Chk2 Antibody #2662 detects endogenous levels of total Chk2.

Western Blotting

Western Blotting

Western blot analysis of extracts from UV or HU treated Mv1Lu cells, using Phospho-Chk1 (Ser317) Antibody #2344.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa, COS, NIH/3T3 and C6 cells, untreated or UV-treated, using Phospho-Chk1 (Ser345) (133D30) Rabbit mAb #2348.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Chk1 (2G1D5) Mouse mAb #2360.


Western Blotting

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 Blotting

Western blot analysis of extracts from GM536, C6, and M059J cells, using Chk2 Antibody #2662.

Western Blotting

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 Blotting

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

Western Blotting

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 Blotting

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


Western Blotting

Western Blotting

Western blot analysis of extracts from 293 cells, untreated or UV-treated (50 mJ/cm2, 2hrs), using Phospho-Chk2 (Ser516) Antibody #2669 (upper) or Chk2 Antibody #2662 (lower).

IHC-P (paraffin)

IHC-P (paraffin)

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

IF-IC

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).


Western blot analysis of extracts from Hela and Cos cells, untreated or treated with 100 mJ/cm2 UV light with 1 hour recovery, using Phospho-Chk1 (Ser296) Antibody, #2349.

Description

The Phospho-Chk1/2 Antibody Sampler Kit offers an economical means to evaluate the phosphorylation status of Chk1 and Chk2 on multiple residues. The kit contains enough primary and secondary antibodies to perform four Western blot experiments with each primary antibody.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser345 of human Chk1. Polyclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Ser296 or Ser317 of human Chk1 and Ser19, Ser33/35, Thr68, Ser516 of human Chk2. Chk1 (2G1D5) Mouse mAb #2360 is produced by immunizing animals with purified recombinant Chk1 protein. Chk 2 Antibody #2662 is produced by immunizing rabbits with a synthetic peptide 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 and plays an important role in DNA damage checkpoint control, embryonic development, and tumor suppression (1). Activation of Chk1 involves phosphorylation at Ser317 and Ser345 and occurs in response to blocked DNA replication and certain forms of genotoxic stress (2). While phosphorylation at Ser345 serves to localize Chk1 to the nucleus following checkpoint activation (3), phosphorylation at Ser317 along with site-specific phosphorylation of PTEN allows for re-entry into the cell cycle following stalled DNA replication (4). Chk1 exerts its checkpoint mechanism on the cell cycle, in part, by regulating the cdc25 family of phosphatases. Chk1 phosphorylation of cdc25A targets it for proteolysis and inhibits its activity through 14-3-3 binding (5). Activated Chk1 can inactivate cdc25C via phosphorylation at Ser216, blocking the activation of cdc2 and transition into mitosis (6). Centrosomal Chk1 has been shown to phosphorylate cdc25B and inhibit its activation of CDK1-cyclin B1, thereby abrogating mitotic spindle formation and chromatin condensation (7). Furthermore, Chk1 plays a role in spindle checkpoint function through regulation of aurora B and BubR1 (8). Research studies have implicated Chk1 as a drug target for cancer therapy as its inhibition leads to cell death in many cancer cell lines (9).

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. Liu, Q. et al. (2000) Genes Dev 14, 1448-59.
  2. Zhao, H. and Piwnica-Worms, H. (2001) Mol Cell Biol 21, 4129-39.
  3. Jiang, K. et al. (2003) J Biol Chem 278, 25207-17.
  4. Martin, S.A. and Ouchi, T. (2008) Mol Cancer Ther 7, 2509-16.
  5. Chen, M.S. et al. (2003) Mol Cell Biol 23, 7488-97.
  6. Zeng, Y. et al. (1998) Nature 395, 507-10.
  7. Löffler, H. et al. (2006) Cell Cycle 5, 2543-7.
  8. Zachos, G. et al. (2007) Dev Cell 12, 247-60.
  9. Garber, K. (2005) J Natl Cancer Inst 97, 1026-8.
  10. Allen, J. B. et al. (1994) Genes Dev. 8, 2401-2415.
  11. Weinert, T. A. et al. (1994) Genes Dev. 8, 652-665.
  12. Murakami, H. and Okayama, H. (1995) Nature 374, 817-819.
  13. Kastan, M.B. and Lim, D.S. (2000) Nat. Rev. Mol. Cell Biol. 1, 179-186.
  14. Matsuoka, S. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 10389-10394.
  15. Melchionna, R. et al. (2000) Nat. Cell Biol. 2, 762-765.
  16. Ahn, J. Y. et al. (2000) Cancer Res. 60, 5934-5936.

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