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8342
UV Induced DNA Damage Response Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

UV Induced DNA Damage Response Antibody Sampler Kit #8342

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UV Induced DNA Damage Response Antibody Sampler Kit: Image 1

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.

UV Induced DNA Damage Response Antibody Sampler Kit: Image 2

Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using Phospho-cdc25C (Ser216) (63F9) Rabbit mAb.

UV Induced DNA Damage Response Antibody Sampler Kit: Image 3

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.

UV Induced DNA Damage Response Antibody Sampler Kit: Image 4

Confocal immunofluorescent analysis of HeLa cells, untreated (left) or UV-treated (right), using RPA32 (4E4) Rat mAb (green) showing translocation to distinct nuclear foci after UV damage. Actin filaments have been labeled with Alexa Fluor® 555 phalloidin. Blue pseudocolor = DRAQ5™ (fluorescent DNA dye).

UV Induced DNA Damage Response Antibody Sampler Kit: Image 5

Confocal immunofluorescent analysis of HeLa cells using ATRIP Antibody (green). Actin filaments have been labeled with Alexa Fluor®555 phalloidin (red).

UV Induced DNA Damage Response Antibody Sampler Kit: Image 6

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.

UV Induced DNA Damage Response Antibody Sampler Kit: Image 7

Western blot analysis of extracts from various cell types using Microcephalin-1/BRIT1 (D38G5) Rabbit mAb.

UV Induced DNA Damage Response 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.

UV Induced DNA Damage Response Antibody Sampler Kit: Image 9

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.

UV Induced DNA Damage Response Antibody Sampler Kit: Image 10

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).

UV Induced DNA Damage Response Antibody Sampler Kit: Image 11

Immunohistochemical analysis of paraffin-embedded human colon carcinoma, showing cytoplasmic localization, using Phospho-cdc25C (Ser216) (63F9) Rabbit mAb.

UV Induced DNA Damage Response Antibody Sampler Kit: Image 12

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).

UV Induced DNA Damage Response Antibody Sampler Kit: Image 13

Western blot analysis of extracts from various cell lines, using RPA32 (4E4) Rat mAb.

UV Induced DNA Damage Response Antibody Sampler Kit: Image 14

Western blot analysis of extracts from Jurkat and HEL cells using ATRIP Antibody.

UV Induced DNA Damage Response Antibody Sampler Kit: Image 15

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).

UV Induced DNA Damage Response 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.

UV Induced DNA Damage Response Antibody Sampler Kit: Image 17

Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using Phospho-cdc25C (Ser216) (63F9) Rabbit mAb.

UV Induced DNA Damage Response Antibody Sampler Kit: Image 18

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

UV Induced DNA Damage Response 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).

UV Induced DNA Damage Response Antibody Sampler Kit: Image 20

Immunohistochemical analysis of paraffin-embedded human lung carcinoma, untreated (left) or lambda phosphatase-treated (right), using Phospho-cdc25C (Ser216) (63F9) Rabbit mAb.

UV Induced DNA Damage Response Antibody Sampler Kit: Image 21

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

UV Induced DNA Damage Response Antibody Sampler Kit: Image 22

Immunohistochemical analysis of paraffin-embedded human lymph node, using Phospho-cdc25C (Ser216) (63F9) Rabbit mAb in the presence of control peptide (left) or Phospho-cdc25C (Ser216) Blocking Peptide #1190 (right).

UV Induced DNA Damage Response 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.

UV Induced DNA Damage Response Antibody Sampler Kit: Image 24

Western Blot analysis of HT29 cells, untreated, nocodazole-treated, and lambda phosphotase-treated, using Phospho-cdc25C (Ser216) antibody (upper), and cdc25C (5H9) Rabbit Mab, #4688, (lower).

UV Induced DNA Damage Response 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.

UV Induced DNA Damage Response Antibody Sampler Kit: Image 26

Western blot analysis of GST-cdc25C, unphosphorylated or phosphorylated in vitro by Chk2, using Phospho-cdc25C (Ser216) (63F9) Rabbit mAb (upper) or cdc25C antibody #9522 (lower).

UV Induced DNA Damage Response Antibody Sampler Kit: Image 27

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 # 8342T
Product # Size Price
8342T
1 Kit  (7 x 20 µl) N/A

Product Description

The UV Induced DNA Damage Response Antibody Sampler Kit offers an economical means of investigating proteins involved in the cellular response to UV-induced DNA damage. The kit contains enough primary and secondary antibody to perform two western blot experiments per primary.

Specificity / Sensitivity

Antibodies detect endogenous levels of the respective target proteins.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly267 of human ATRIP protein or Ser428 of human ATR protein. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Monoclonal antibodies are produced by immunizing animals with a recombinant full-length human Maltose Binding Protein-RPA32 fusion protein or a synthetic peptide corresponding to residues surrounding Tyr415 of human Microcephalin-1/BRIT1 protein. Activation state monoclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser139 of human H2A.X protein, Ser216 of human cdc25C protein, or Ser345 of human Chk1 protein, respectively.

Background

Exposure to ultraviolet radiation (UV) has a profound impact on human health and disease (1). Low level UV exposure induces the production of vitamin D and is a key regulator of calcium metabolism. Conversely, overexposure to UV is associated with an increased risk of cancer, immunosuppression, and many eye disorders, such as cataracts. Photons of UV light can directly damage DNA causing thymine dimers and other pyrimidine dimers between adjacent bases (2). Free radicals and reactive oxygen species induced by UV exposure also result in DNA lesions and have been linked to malignant melanoma (3). DNA damage from replicative stress and genotoxic agents like UV activate the ATR-mediated checkpoint pathway and stimulate DNA repair, cell cycle arrest, and apoptosis (4). ATR recruitment to sites of DNA damage and activation depends, at least in part, on interaction with the complex of single-stranded DNA, Replication Protein A (RPA), and direct binding to the ATR-associated adapter protein, ATRIP (5). In addition, the Rad17-RFC and Rad9-Rad1-Hus1 (9-1-1) protein complexes are independently recruited with TopBP1 to fully activate the checkpoint response (6,7). BRIT1 (MCPH1) is required for UV-induced formation of ATR, RPA, and p-Rad17 foci at sites of DNA damage (8-10) and may regulate the expression of several DNA damage response proteins (11). Once activated, ATR phosphorylates a number of mediators, including histone H2AX Ser139 and Chk1 kinase at Ser345. H2AX phosphorylation is a marker of DNA damage. Complete loss of H2AX results in reduced Chk1 activation and impaired survival of cells after UV exposure (12). Chk1 and Chk2 kinase activation is essential for checkpoint-mediated control of cell cycle progression (4). Checkpoint kinases stimulate cell cycle arrest by phosphorylation of a group of tyrosine phosphatases known as Cdc25A, Cdc25B, and Cdc25C (13 -15). Both Chk1 and Chk2 kinases phosphorylate Cdc25C at Ser216 in response to DNA damage and stimulate arrest (16-17).

  1. von Thaler, A.K. et al. (2010) Exp Dermatol 19, 81-8.
  2. Rastogi, R.P. et al. (2010) J Nucleic Acids 2010, 592980.
  3. Narayanan, D.L. et al. (2010) Int J Dermatol 49, 978-86.
  4. Zhou, B.B. and Elledge, S.J. (2000) Nature 408, 433-9.
  5. Zou, L. and Elledge, S.J. (2003) Science 300, 1542-8.
  6. Zou, L. et al. (2002) Genes Dev 16, 198-208.
  7. Mordes, D.A. and Cortez, D. (2008) Cell Cycle 7, 2809-12.
  8. Rai, R. et al. (2006) Cancer Cell 10, 145-57.
  9. Peng G et al. (2009) Nat Cell Biol 11, 865–72
  10. Lin, S.Y. et al. (2010) Yonsei Med J 51, 295-301.
  11. Lin, S.Y. et al. (2005) Proc Natl Acad Sci U S A 102, 15105-9.
  12. Revet, I. et al. (2011) Proc Natl Acad Sci U S A 108, 8663-7.
  13. Mailand, N. et al. (2000) Science 288, 1425-9.
  14. Sanchez, Y. et al. (1997) Science 277, 1497-501.
  15. Matsuoka, S. et al. (1998) Science 282, 1893-7.
  16. Blasina, A. et al. (1999) Curr. Biol. 9, 1-10.
  17. Furnari, B. et al. (1999) Mol. Biol. Cell 10, 833-845.

Limited Uses

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