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9947
DNA Damage Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

DNA Damage Antibody Sampler Kit #9947

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DNA Damage Antibody Sampler Kit: Image 1

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.

DNA Damage Antibody Sampler Kit: Image 2

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).

DNA Damage Antibody Sampler Kit: Image 3

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.

DNA Damage Antibody Sampler Kit: Image 4

Flow cytometric analysis of HeLa cells, untreated (blue) and UV-treated (100 mJ/cm2, 1 hr recovery; green), using Phospho-Chk1 (Ser345) (133D3) Rabbit mAb.

DNA Damage Antibody Sampler Kit: Image 5

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.

DNA Damage Antibody Sampler Kit: Image 6

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).

DNA Damage Antibody Sampler Kit: Image 7

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).

DNA Damage Antibody Sampler Kit: Image 8

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.

DNA Damage Antibody Sampler Kit: Image 9

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).

DNA Damage Antibody Sampler Kit: Image 10

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

DNA Damage Antibody Sampler Kit: Image 11

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).

DNA Damage Antibody Sampler Kit: Image 12

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).

DNA Damage Antibody Sampler Kit: Image 13

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).

DNA Damage Antibody Sampler Kit: Image 14

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

DNA Damage Antibody Sampler Kit: Image 15

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

DNA Damage Antibody Sampler Kit: Image 16

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

DNA Damage Antibody Sampler Kit: Image 17

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).

DNA Damage Antibody Sampler Kit: Image 18

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

DNA Damage Antibody Sampler Kit: Image 19

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).

DNA Damage Antibody Sampler Kit: Image 20

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

DNA Damage Antibody Sampler Kit: Image 21

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

DNA Damage Antibody Sampler Kit: Image 22

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

DNA Damage Antibody Sampler Kit: Image 23

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

DNA Damage Antibody Sampler Kit: Image 24

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

DNA Damage Antibody Sampler Kit: Image 25

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

DNA Damage Antibody Sampler Kit: Image 26

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) N/A

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) Nature Rev. Mol. Cell Biol. 1, 179-186.
  2. Abraham, R.T. (2004) DNA Repair (Amst) 3, 883-7.
  3. Shechter, D. et al. (2004) DNA Repair (Amst) 3, 901-8.
  4. Shieh, S.Y. et al. (1997) Cell 91, 325-34.
  5. Tibbetts, R.S. et al. (1999) Genes Dev 13, 152-7.
  6. Martinho, R.G. et al. (1998) EMBO J. 17, 7239-17249.
  7. Matsuoka, S. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 10389-10394.
  8. Melchionna, R. et al. (2000) Nat. Cell Biol. 2, 762-765.
  9. Ahn, J.Y. et al. (2000) Cancer Res. 60, 5934-5936.
  10. Rogakou, E.P. et al. (1998) J Biol Chem 273, 5858-68.
  11. Burma, S. et al. (2001) J Biol Chem 276, 42462-7.
  12. Rogakou, E.P. et al. (1999) J Cell Biol 146, 905-16.

Limited Uses

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Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST's products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST's Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

For Research Use Only. Not For Use In Diagnostic Procedures.
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