Revision 1

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Orders:

877-616-CELL (2355)

[email protected]

Support:

877-678-TECH (8324)

3 Trask Lane | Danvers | Massachusetts | 01923 | USA

For Research Use Only. Not for Use in Diagnostic Procedures.

Product Includes Product # Quantity Mol. Wt Isotype/Source
Phospho-Histone H2A.X (Ser139) (20E3) Rabbit Monoclonal Antibody971820 µl15 kDaRabbit IgG
Phospho-cdc25C (Ser216) (63F9) Rabbit Monoclonal Antibody490120 µl60 kDaRabbit IgG
Phospho-Chk1 (Ser345) (133D3) Rabbit Monoclonal Antibody234820 µl56 kDaRabbit IgG
RPA32/RPA2 (4E4) Rat Monoclonal Antibody220820 µl32 kDaRat IgG1
ATRIP Antibody273720 µl82 kDaRabbit
Phospho-ATR (Ser428) Antibody285320 µl300 kDaRabbit
Microcephalin-1/BRIT1 (D38G5) Rabbit Monoclonal Antibody412020 µl100 kDaRabbit IgG
Anti-rabbit IgG, HRP-linked Antibody7074100 µlGoat
Anti-rat IgG, HRP-linked Antibody7077100 µlGoat

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

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.

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

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).

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

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 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 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.

Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

Cell Signaling Technology Logo
Western blot analysis of extracts from various cell lines, using RPA32 (4E4) Rat mAb.
undefined Image 1: UV Induced DNA Damage Response Antibody Sampler Kit
Flow cytometric analysis of Jurkat cells using RPA32/RPA2 (4E4) Rat mAb (right) and DAPI #4083, compared to concentration-matched Rat IgG1 isotype control (left). Anti-rat IgG (H+L), (Alexa Fluor® 647 Conjugate) #4418 was used as a secondary antibody.
undefined Image 2: UV Induced DNA Damage Response Antibody Sampler Kit
Western blot analysis of extracts from HeLa, COS, NIH/3T3 and C6 cells, untreated or UV-treated, using Phospho-Chk1 (Ser345) (133D30) Rabbit mAb.
undefined Image 3: UV Induced DNA Damage Response Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

Cell Signaling Technology Logo
Western blot analysis of extracts from Jurkat and HEL cells using ATRIP Antibody.
undefined Image 4: UV Induced DNA Damage Response Antibody Sampler Kit
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.
undefined Image 5: UV Induced DNA Damage Response Antibody Sampler Kit
Western blot analysis of extracts from various cell types using Microcephalin-1/BRIT1 (D38G5) Rabbit mAb.
undefined Image 6: UV Induced DNA Damage Response Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

Cell Signaling Technology Logo
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).
undefined Image 7: UV Induced DNA Damage Response Antibody Sampler Kit
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.
undefined Image 8: UV Induced DNA Damage Response Antibody Sampler Kit
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.
undefined Image 9: UV Induced DNA Damage Response Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

Cell Signaling Technology Logo
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).
undefined Image 10: UV Induced DNA Damage Response Antibody Sampler Kit
Immunohistochemical analysis of paraffin-embedded human ovarian clear cell carcinoma using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb performed on the Leica BOND Rx.
undefined Image 11: UV Induced DNA Damage Response Antibody Sampler Kit
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).
undefined Image 12: UV Induced DNA Damage Response Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

Cell Signaling Technology Logo
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).
undefined Image 13: UV Induced DNA Damage Response Antibody Sampler Kit
Confocal immunofluorescent analysis of HeLa cells using ATRIP Antibody (green). Actin filaments have been labeled with Alexa Fluor®555 phalloidin (red).
undefined Image 14: UV Induced DNA Damage Response Antibody Sampler Kit
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).
undefined Image 15: UV Induced DNA Damage Response Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

Cell Signaling Technology Logo
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).
undefined Image 16: UV Induced DNA Damage Response Antibody Sampler Kit
Immunohistochemical analysis of paraffin-embedded human prostate adenocarcinoma using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb performed on the Leica BOND Rx.
undefined Image 17: UV Induced DNA Damage Response Antibody Sampler Kit
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.
undefined Image 18: UV Induced DNA Damage Response Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

Cell Signaling Technology Logo
Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using Phospho-cdc25C (Ser216) (63F9) Rabbit mAb.
undefined Image 19: UV Induced DNA Damage Response Antibody Sampler Kit
Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb, showing nuclear localization.
undefined Image 20: UV Induced DNA Damage Response Antibody Sampler Kit
Immunohistochemical analysis of paraffin-embedded human colon carcinoma, showing cytoplasmic localization, using Phospho-cdc25C (Ser216) (63F9) Rabbit mAb.
undefined Image 21: UV Induced DNA Damage Response Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

Cell Signaling Technology Logo
Immunohistochemical analysis of paraffin-embedded human colon carcinoma, using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb.
undefined Image 22: UV Induced DNA Damage Response Antibody Sampler Kit
Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using Phospho-cdc25C (Ser216) (63F9) Rabbit mAb.
undefined Image 23: UV Induced DNA Damage Response Antibody Sampler Kit
Immunohistochemical analysis of paraffin-embedded human lung carcinoma untreated (left) or lambda-phosphatase-treated (right), using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb.
undefined Image 24: UV Induced DNA Damage Response Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

Cell Signaling Technology Logo
Immunohistochemical analysis of paraffin-embedded human lung carcinoma, untreated (left) or lambda phosphatase-treated (right), using Phospho-cdc25C (Ser216) (63F9) Rabbit mAb.
undefined Image 25: UV Induced DNA Damage Response Antibody Sampler Kit
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).
undefined Image 26: UV Induced DNA Damage Response Antibody Sampler Kit
Immunohistochemical analysis of paraffin-embedded HT-29 cells untreated (left) or UV-treated (right), using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb.
undefined Image 27: UV Induced DNA Damage Response Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

Cell Signaling Technology Logo
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).
undefined Image 28: UV Induced DNA Damage Response Antibody Sampler Kit
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.
undefined Image 29: UV Induced DNA Damage Response Antibody Sampler Kit
Immunoprecipitation of RPA32/RPA2 protein from HeLa cell extracts. Lane 1 is 10% input and lane 2 is RPA32/RPA2 (4E4) Rat mAb. Western blot analysis was performed using RPA32/RPA2 (E8X5P) XP® Rabbit mAb #35869.
undefined Image 30: UV Induced DNA Damage Response Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

Cell Signaling Technology Logo
Simple Western™ analysis of lysates (0.1 mg/mL) from HeLa cells treated with UV (100mJ/cm2; 2 hour recovery) using Phospho-Chk1 (Ser345) (133D3) Rabbit mAb #2348. The virtual lane view (left) shows a single target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ ​​​​​​​ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
undefined Image 31: UV Induced DNA Damage Response Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.