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9947
DNA Damage Antibody Sampler Kit
Primary Antibodies

DNA Damage Antibody Sampler Kit #9947

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Western blot analysis of Raw264.7, SV-T2 and HT-29 cells that were untreated or UV-treated (50 mJ, 30 min), using Phospho-ATR (Ser428) Antibody. Lambda phosphatase NEB #P0753 (10,000 Units/ml, 1hr) was used to demonstrate the phospho-specificity of the antibody.

Western blot analysis of untreated and UV-treated (50 mJ/cm2,30 min) HeLa cells and HT-1376 cells, using Phospho-BRCA1 (Ser1524) Antibody (upper) and BRCA1 Antibody #9010 (lower).

Flow cytometric analysis of HeLa cells, untreated (blue) or treated with UV (100mJ/cm2, 2 hr recovery; green) using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) was used as a secondary antibody.

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

Flow cytometric analysis of HeLa cells, untreated (blue) or treated with UV (100 mJ, 2hr recovery; green) using Phospho-H2A.X (Ser139) (20E3) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® isotype control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

Flow cytometric analysis of HT-29 cells, untreated (blue) or UV-treated (green), using Phospho-p53 (Ser15) (16G8) Mouse mAb compared to a nonspecific negative control antibody (red).

Western blot analysis of extracts from 293 cells, untreated or UV-treated (100 mJ, 4 hr recovery), using Phospho-ATM (Ser1981) (D6H9) Rabbit mAb (upper) or ATM (D2E2) Rabbit mAb #2873 (lower).

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.

Western blot analysis of untreated, UV-treated (50 mJ, 30 min) and nocodazole-treated (50 ng/ml, 24hr) Raw264.7 cells, using Phospho-ATR (Ser428) Antibody (upper) and a total ATR antibody (lower).

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

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).

Confocal immunofluorescent analysis of HeLa cells, untreated (left) or UV-treated (right), using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).

Confocal immunofluorescent analysis of HT-29 cells, untreated (left) or UV-treated (right), using Phospho-p53 (Ser15) (16G8) Mouse mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red).

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

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

Immunohistochemical analysis of paraffin-embedded HT-29 cells untreated (left) or UV-treated (right), using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb.

Western blot analysis of extracts from HT29 cells, untreated or UV-treated (100 mJ/cm2, 1 hr), using Phospho-p53 (Ser15) (16G8) Mouse mAb (upper) or p53 (DO-7) Mouse mAb #48818 (lower).

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

Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb in the presence of control peptide (left) or Phospho-Histone H2A.X (Ser139) Blocking Peptide #1260 (right).

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

Immunohistochemical analysis of paraffin-embedded human colon carcinoma, using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb.

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

Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb, showing nuclear localization.

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

Immunohistochemical analysis of paraffin-embedded human lung carcinoma untreated (left) or lambda-phosphatase-treated (right), using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb.

Western blot analysis of extracts from untreated or UV-treated 293 cells, using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (upper) or Histone H2A.X Antibody #2595 (lower).

To Purchase # 9947T
Product # Size Price
9947T
1 Kit  (7 x 20 µl) $ 485

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-ATR (Ser428) Antibody 2853 20 µl
  • WB
H M R Mk 300 Rabbit 
Phospho-BRCA1 (Ser1524) Antibody 9009 20 µl
  • WB
H 220 Rabbit 
Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb 2197 20 µl
  • WB
  • IP
  • IHC
  • F
H 62 Rabbit IgG
Phospho-Chk1 (Ser345) (133D3) Rabbit mAb 2348 20 µl
  • WB
  • IF
  • F
H M R Mk 56 Rabbit IgG
Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb 9718 20 µl
  • WB
  • IHC
  • IF
  • F
H M R Mk 15 Rabbit IgG
Phospho-p53 (Ser15) (16G8) Mouse mAb 9286 20 µl
  • WB
  • IF
  • F
H 53 Mouse IgG1
Phospho-ATM (Ser1981) (D6H9) Rabbit mAb 5883 20 µl
  • WB
H 350 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 
Anti-mouse IgG, HRP-linked Antibody 7076 100 µl
  • WB
Horse 

Product Description

This kit provides an economical means to analyze major signaling checkpoints in response to DNA damage. The kit contains primary and secondary antibodies to perform two Western blots with each antibody.

Specificity / Sensitivity

All antibodies in the DNA Damage Antibody Sampler Kit recognize their targets proteins only when modified at the indicated site.

Source / Purification

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.

Background

Ataxia telangiectasia mutated kinase (ATM) and ataxia telangiectasia and Rad3-related kinase (ATR) are PI3 Kinase-related kinase (PIKK) family members that phosphorylate multiple substrates on serine or threonine residues that are followed by a glutamine in response to DNA damage or replication blocks (1-3). p53 is phosphorylated by ATM, ATR and DNA-PK at Ser15. This phosphorylation impairs the ability of MDM2 to bind p53, promoting both the accumulation and activation of p53 in response to DNA damage (4,5). Chk1 and Chk2, downstream protein kinases of ATM/ATR, plays an important role in DNA damage checkpoint control, embryonic development and tumor suppression (6). Chk1 is phosphorylated at Ser280 and Ser296 following DNA damage. The amino-terminal domain of Chk2 contains a series of seven serine or threonine residues, including Thr68, each followed by glutamine (SQ or TQ motif). After DNA damage by ionizing radiation (IR), UV irradiation or hydroxyurea treatment, Thr68 and other sites in this region become phosphorylated by ATM/ATR (7-9). The breast cancer susceptibility proteins BRCA1 and BRCA2 are frequently mutated in cases of hereditary breast and ovarian cancers and have roles in multiple processes related to DNA damage, repair, cell cycle progression, transcription, ubiquitination and apoptosis. Numerous DNA-damage induced phosphorylation sites on BRCA1 have been identified, including serine 1524, and kinases activated in a cell cycle-dependent manner, including Aurora A and CDK2, can also phosphorylate BRCA1. IR, DNA and radiometric-induced DNA damage also results in rapid phosphorylation of the histone H2A family member H2A.X at Ser139 by ATM (10,11). Within minutes following DNA damage, Ser139-phosphorylated H2A.X localizes to sites of DNA damage at subnuclear foci (12).

  1. Kastan, M.B. and Lim, D.S. (2000) Nat. Rev. Mol. Cell Biol. 1, 179-186.
  2. Abraham, R.T. DNA Repair (Amst) 3, 883-887.
  3. Rogakou, E.P. et al. (1998) J. Biol. Chem. 273, 5858-5868.
  4. Shechter, D. et al. DNA Repair (Amst) 3, 901-908.
  5. Burma, S. et al. (2001) J. Biol. Chem. 276, 42462-42467.
  6. Shieh, S.Y. et al. (1997) Cell 91, 325-34.
  7. Rogakou, E.P. et al. (1999) J. Cell Biol. 146, 905-916.
  8. Tibbetts, R.S. et al. (1999) Genes Dev 13, 152-7.
  9. Martinho, R.G. et al. (1998) EMBO J. 17, 7239-17249.
  10. Matsuoka, S. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 10389-10394.
  11. Melchionna, R. et al. (2000) Nat. Cell Biol. 2, 762-765.
  12. Ahn, J.Y. et al. (2000) Cancer Res. 60, 5934-5936.
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. 7,429,487, foreign equivalents, and child patents deriving therefrom.