Revision 4

#9947

Store at -20C

DNA Damage Antibody Sampler Kit

1 Kit

(7 x 20 microliters)

CST Logo
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-ATR (Ser428) Antibody285320 µl300 kDaRabbit
Phospho-BRCA1 (Ser1524) Antibody900920 µl220 kDaRabbit
Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb219720 µl62 kDaRabbit IgG
Phospho-Chk1 (Ser345) (133D3) Rabbit mAb234820 µl56 kDaRabbit IgG
Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb971820 µl15 kDaRabbit IgG
Phospho-p53 (Ser15) (16G8) Mouse mAb928620 µl53 kDaMouse IgG1
Phospho-ATM (Ser1981) (D25E5) Rabbit mAb1305020 µl350 kDaRabbit IgG
Anti-rabbit IgG, HRP-linked Antibody7074100 µlGoat
Anti-mouse IgG, HRP-linked Antibody7076100 µlHorse

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

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.

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

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).

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 4

Cell Signaling Technology Logo
Western blot analysis of extracts from HCT 116 cells, untreated (-) or treated with neocarzinostatin (NCS 10 μM, 1 hr; +), using Phospho-ATM (Ser1981) (D25E5) Rabbit mAb (upper) and ATM (D2E2) Rabbit mAb #2873 (lower).
undefined Image 1: DNA Damage Antibody Sampler Kit
Western blot analysis of extracts from HeLa cells, untreated or UV-treated, using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb.
undefined Image 2: DNA Damage 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: DNA Damage 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 4

Cell Signaling Technology Logo
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 4: DNA Damage 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 5: DNA Damage 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 6: DNA Damage 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 4

Cell Signaling Technology Logo
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).
undefined Image 7: DNA Damage Antibody Sampler Kit
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).
undefined Image 8: DNA Damage Antibody Sampler Kit
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 9: DNA Damage 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 4

Cell Signaling Technology Logo
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 10: DNA Damage Antibody Sampler Kit
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 11: DNA Damage 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 12: DNA Damage 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 4

Cell Signaling Technology Logo
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 13: DNA Damage Antibody Sampler Kit
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb.
undefined Image 14: DNA Damage 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 15: DNA Damage 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 4

Cell Signaling Technology Logo
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).
undefined Image 16: DNA Damage 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 17: DNA Damage Antibody Sampler Kit
Immunohistochemical analysis of paraffin-embedded human colon carcinoma, control (left) or λ phosphatase-treated (right), using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb.
undefined Image 18: DNA Damage 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 4

Cell Signaling Technology Logo
Immunohistochemical analysis of paraffin-embedded human colon carcinoma, using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb.
undefined Image 19: DNA Damage Antibody Sampler Kit
Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb.
undefined Image 20: DNA Damage 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 21: DNA Damage 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 4

Cell Signaling Technology Logo
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 22: DNA Damage Antibody Sampler Kit
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 23: DNA Damage 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 24: DNA Damage 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 4

Cell Signaling Technology Logo
Immunohistochemical analysis of paraffin-embedded HCT 116 cell pellets, control (top) or CRISPR/Cas9 Chk2 knockout (KO) (bottom), untreated (left) or UV-treated (100 mJ, 2 hr recovery, right) using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb.
undefined Image 25: DNA Damage Antibody Sampler Kit

Simple Western™ analysis of lysates (0.1 mg/mL) from 293 cells treated with UV (100 mJ/cm2; 2 Hour Recovery) using Phospho-ATM (Ser1981) (D25E5) Rabbit mAb #13050. The virtual lane view (left) shows the 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 66 - 440 kDa separation module.

undefined Image 26: DNA Damage Antibody Sampler Kit
Simple Western™ analysis of lysates (0.1 mg/mL) from HEK 293 cells treated with UV (50 mJ, 30 min recovery) using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb #2197. The virtual lane view (left) shows a single target band (as indicated) at 1:50 and 1:250 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:50 (blue line) and 1:250 (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 27: DNA Damage 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 4

Cell Signaling Technology Logo
Immunoprecipitation of Phospho-Chk2 (Thr68) from HeLa cell extracts. Cells were treated with Staurosporine (1uM, 3hrs.) Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb. Western blot analysis was performed using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678 was used as a secondary antibody.
undefined Image 28: DNA Damage Antibody Sampler Kit
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 29: DNA Damage Antibody Sampler Kit
Flow cytometric analysis of HT-29 cells, untreated (blue) or treated with UV (100 mJ/cm2, 2hr recovery; green) using Phospho-p53 (Ser15) (16G8) Mouse mAb (solid lines) or concentration-matched Mouse (G3A1) mAb IgG1 Isotype Control #5415 (dashed lines). Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4408 was used as a secondary antibody.
undefined Image 30: DNA Damage 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.