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9931
Phospho-Chk1/2 Antibody Sampler Kit
Primary Antibodies

Phospho-Chk1/2 Antibody Sampler Kit #9931

 Image 1

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.

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Western Blotting Image 2

Western blot analysis of extracts from 293 and NIH/3T3 cells, untreated (-) or UV-treated (100 mJ, 1 hr recovery; +), using Phospho-Chk1 (Ser317) (D12H3) XP® Rabbit mAb. The blot on the right was treated with calf intestinal phosphatase (CIP) before western blot.

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Western Blotting Image 3

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

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Western Blotting Image 4

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.

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Western Blotting Image 5

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

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Western Blotting Image 6

Western blot analysis of extracts from various cell lines using Chk2 (D9C6) XP® Rabbit mAb.

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Western Blotting Image 7

Western blot analysis of extracts from COS cells, untransfected (lane 1) or transfected with Wild-type Chk2 (lane 2), Chk2 (S19A) (lane 3), Chk2 (T26S28A) (lane 4), Chk2 (S33S35A) (lane 5) or Chk2 (T68A) (lane 6), using Phospho-Chk2 (Ser19) Antibody.

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Western Blotting Image 8

Western blot analysis of extracts from COS cells, untransfected (lane 1) or transfected with Wild-type Chk2 (lane 2), Chk2 (S19A) (lane 3), Chk2 (T26S28A) (lane 4), Chk2 (S33S35A) (lane 5) or Chk2 (T68A) (lane 6), using Phospho-Chk2 (Ser33/35) Antibody.

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Western Blotting Image 9

Western blot analysis of extracts from HeLa cells, untreated or UV-treated, using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb.

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Western Blotting Image 10

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

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Western Blotting Image 11

After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.

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IP Image 12

Immunoprecipitation of phospho-Chk1 (Ser317) from 293 cell extracts treated with UV (100 mJ, 1 hr recovery) using Phospho-Chk1 (Ser317) (D12H3) XP® Rabbit mAb (lane 2) or Rabbit (D1AG) mAb IgG XP® Isotype Control #3900 (lane 3). Lane 1 is 10% input.

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Flow Cytometry Image 13

Flow cytometric analysis of HeLa cells, untreated (blue) and UV-treated (green), using Phospho-Chk1 (Ser345) (133D3) Rabbit mAb.

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Western Blotting Image 14

Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 (-) or SignalSilence® Chk1 siRNA I #6241 or SignalSilence® Chk1 siRNA II (+), using Chk1 (2G1D5) Mouse mAb #2360 and β-Actin (13E5) Rabbit mAb #4970. Chk1 (2G1D5) Mouse mAb confirms silencing of Chk1 expression and β-Actin (13E5) Rabbit mAb is used to control for loading and specificity of Chk1 siRNA.

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IP Image 15

Immunoprecipitation of Chk2 from 293 cell extracts using Chk2 (D9C6) XP® Rabbit mAb (lane 2). Western blot detection was performed using the same antibody. Lane 1 is 10% input.

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Western Blotting Image 16

Western blot analysis of extracts from HeLa cells treated with UV for the indicated times, using Phospho-Chk2 (Ser19) Antibody.

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Western Blotting Image 17

Western blot analysis of extracts from HeLa cells treated with UV for the indicated times, using Phospho-Chk2 (Ser33/35) Antibody.

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IP Image 18

Immunoprecipitation of phospho-chk2 from UV-treated HT29 cells using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb followed by western blot using the same antibody.

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IF-IC Image 19

Confocal immunofluorescent analysis of HeLa cells, untreated (left), UV-treated (center), or UV and λ phosphatase-treated (right), using Phospho-Chk1 (Ser317) (D12H3) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).

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IF-IC Image 20

Confocal immunofluorescent analysis of C2C12 cells, untreated (left) or UV-treated (right), using Phospho-Chk1 (Ser345) (133D3) Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).

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IHC-P (paraffin) Image 21

Immunohistochemical analysis of paraffin-embedded human lung carcioma using Chk2 (D9C6) XP® Rabbit mAb.

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IHC-P (paraffin) Image 22

Immunohistochemical analysis of paraffin-embedded human breast carcinoma, showing nuclear localization, using Phospho-Chk2 (Ser19) Antibody.

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IHC-P (paraffin) Image 23

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb.

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IHC-P (paraffin) Image 24

Immunohistochemical analysis of paraffin-embedded human ovarian serous adenocarcinoma using Chk2 (D9C6) XP® Rabbit mAb.

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IHC-P (paraffin) Image 25

Immunohistochemical analysis of paraffin-embedded human colon carcinoma, control (left) or λ phosphatase-treated (right), using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb.

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IHC-P (paraffin) Image 26

Immunohistochemical analysis of paraffin-embedded cell pellets, HCT 116 (left) or MDA-MB-231 (right), using Chk2 (D9C6) XP® Rabbit mAb.

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IHC-P (paraffin) Image 27

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb.

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IF-IC Image 28

Confocal immunofluorescent analysis of HCT 116 (left) and HCT-15 (right) cells using Chk2 (D9C6) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).

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IHC-P (paraffin) Image 29

Immunohistochemical analysis of paraffin-embedded HT-29 cell pellets, control (left) or UV-treated (right), using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb.

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Flow Cytometry Image 30

Flow cytometric analysis of untreated Jurkat cells, using Phospho-Chk2 (Thr68) (C13C1) Rb mAb versus propidium iodide (DNA content). The boxed population indicates phospho-Chk2 (Thr68)-positive cells.

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-Chk1 (Ser317) (D12H3) XP® Rabbit mAb 12302 20 µl
  • WB
  • IP
  • IF
H M Mk 56 Rabbit IgG
Phospho-Chk1 (Ser345) (133D3) Rabbit mAb 2348 20 µl
  • WB
  • IF
  • F
H M R Mk 56 Rabbit IgG
Phospho-Chk1 (Ser296) Antibody 2349 20 µl
  • WB
H Mk 56 Rabbit 
Chk1 (2G1D5) Mouse mAb 2360 20 µl
  • WB
H M R Mk 56 Mouse IgG1
Chk2 (D9C6) XP® Rabbit mAb 6334 20 µl
  • WB
  • IP
  • IHC
  • IF
H 62 Rabbit IgG
Phospho-Chk2 (Ser19) Antibody 2666 20 µl
  • WB
  • IHC
H 62 Rabbit 
Phospho-Chk2 (Ser33/35) Antibody 2665 20 µl
  • WB
H Mk 62 Rabbit 
Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb 2197 20 µl
  • WB
  • IP
  • IHC
  • F
H 62 Rabbit IgG
Phospho-Chk2 (Ser516) Antibody 2669 20 µl
  • WB
H 62 Rabbit 
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

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 two Western blot experiments with each primary antibody.

Each antibody in the Phospho-Chk1/2 Antibody Sampler Kit detects endogenous levels of its respective target protein.

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide and are purified by protein A and peptide affinity chromatography. Monoclonal antibodies are produced by immunizing animals with recombinant human proteins or synthetic peptides.

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 by ATM/ATR, followed by autophosphorylation of Ser296. Activation 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. Kastan, M.B. and Lim, D.S. (2000) Nat Rev Mol Cell Biol 1, 179-86.
  12. Weinert, T.A. et al. (1994) Genes Dev. 8, 652-665.
  13. Murakami, H. and Okayama, H. (1995) Nature 374, 817-819.
  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.
Entrez-Gene Id
1111 , 11200
Swiss-Prot Acc.
O14757 , O96017
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
DRAQ5 is a registered trademark of Biostatus Limited.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.

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