Product Pathways - DNA Damage

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Double Strand Breaks (DSB) Repair Antibody Sampler Kit #9653

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IHC-F=Immunohistochemistry (Frozen)  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry  E-P=ELISA (Peptide)
Reactivity Key:  H=Human  M=Mouse  R=Rat  Hm=Hamster  Mk=Monkey  Mi=Mink  B=Bovine  Pg=Pig  Hr=Horse
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Specificity / Sensitivity

Each antibody in the Double Strand Breaks (DSB) Repair Antibody Sampler Kit detects endogenous levels of its respective protein and does not cross-react with other family members. Activation state antibodies only detect their target proteins when modified at the indicated site.

Western Blotting

Western blot analysis of extracts from HeLa, A549 and COS cells using Ku80 (C48E7) Rabbit mAb #2180.

Western Blotting

Western blot analysis of extracts from various cell lines using XLF Antibody #2854.

Western Blotting

Western blot analysis of extracts from Mv 1 Lu cells, untreated, UV-treated, or hydroxyurea (HU) treated for the indicated times, using Phospho-p95/NBS1 (Ser343) Antibody #3001.

Western Blotting

Western blot analysis of extracts from K-562 cells using Rad52 Antibody #3425.

Western Blotting

Western blot analysis of extracts from Jurkat and K-562 cells using RAD50 Antibody #3427.

Western Blotting

Western blot analysis of extracts from wildtype M059K and DNA-PK deficient M059J cells using DNA-PK Antibody #4602.

Western Blotting

Western blot analysis of extracts from HeLa and K-562 cells using Mre11 Rabbit (31H4) mAb #4847.

Western Blotting

Western blot analysis of extracts from untreated or UV-treated (100 mJ, 4 hour recovery) 293 cells using Phospho-ATM (Ser1981) (D6H9) Rabbit mAb #5883 (upper) or ATM (D2E2) Rabbit mAb #2873 (lower).

Western Blotting

Western blot analysis of HeLa and HT-1376 cells, untreated or UV-treated (50 mJ/cm² for 30 min), using Phospho-BRCA1 (Ser1524) Antibody #9009 (upper) and BRCA1 Antibody #9010 (lower).

Description

The Double Strand Breaks (DSB) Repair Antibody Sampler Kit provides an economical means to investigate repair of double-strand DNA breaks within the cell. The kit contains primary and secondary antibodies to perform four western blots with each antibody.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Lys496 of human Mre11A and the carboxy terminus of human Ku80. Activation state monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues around Ser1981 of human ATM. Polyclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to the carboxy-terminus of human DNA-PKcs, the amino terminus of human Rad50, the central sequence of human Rad52, and the carboxy terminus of human XLF. Activation state polyclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Ser1524 of human BRCA1 and surrounding Ser343 of human p95/NBS1. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.

Background

Double strand DNA breaks (DSB) in mammalian cells can be repaired by the related mechanisms of non-homologous end-joining (NHEJ) and homologous recombination (HR). A DNA-dependent protein kinase composed of DNA-binding subunits Ku70 and Ku86 and the DNA-PKcs catalytic subunit mediates NHEJ repair. The Ku heterodimer binds free DNA ends and recruits DNA-PKcs to the break (1). DNA-PKcs signals areas of DNA damage and recruits additional proteins, such as the Artemis exo- and endonuclease that processes and primes the damaged sequence (2,3). Following replacement DNA synthesis, a ligase complex composed of DNA ligase IV and XRCC4 joins the repaired ends. XRCC4-like factor (XLF) is an essential ligase-associated repair factor that promotes gap-filling during NHEJ (4). Homologous recombination utilizes aligned homologous sequences as a repair template. The MRN complex, composed of Mre11, Rad50, and nibrin (p95/NBS1), plays a critical role in sensing, processing and repairing breaks (5). MRN interacts with BRCA1 and CtIP to facilitate 5’ resection of DSB DNA to generate 3’ ssDNA ends necessary for repair (6). DNA-binding protein Mre11 exhibits exonuclease and endonuclease activity and is largely responsible for ssDNA end processing (7). Interaction between the MRN complex and ATM kinase promotes association between the kinase and its substrates and likely leads to ATM activation (8). ATM acts a central controller of the cell cycle checkpoint by phosphorylating multiple targets, including c-Abl, BRCA1 and p95/NSB1. Activated c-Abl phosphorylates Rad52, which promotes Rad51 binding to ssDNA and subsequent annealing of ssDNA (7).

1. Gottlieb, T.M. and Jackson, S.P. (1993) Cell 72, 131-42.
2. Franco, S. et al. (2008) J Exp Med 205, 557-64.
3. Collis, S.J. et al. (2005) Oncogene 24, 949-61.
4. Akopiants, K. et al. (2009) Nucleic Acids Res 37, 4055-62.
5. Williams, R.S. et al. (2007) Biochem Cell Biol 85, 509-20.
6. Chen, L. et al. (2008) J Biol Chem 283, 7713-20.
7. Czornak, K. et al. (2008) J Appl Genet 49, 383-96.
8. Lee, J.H. and Paull, T.T. (2007) Oncogene 26, 7741-8.

Application References

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!

Protocols

2180:

IHC / Frozen, IHC / Paraffin, Immunofluorescence, Immunoprecipitation, Western Blotting

2854:

Immunoprecipitation, Western Blotting

3001:

ELISA Peptide, Western Blotting

3425:

Immunofluorescence, Western Blotting

3427:

Western Blotting

4602:

Western Blotting

4847:

Flow, IHC / Frozen, IHC / Paraffin, Immunoprecipitation, Western Blotting

5883:

Western Blotting

9009:

Western Blotting

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