Revision 3
#12311
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For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:
W, IHC-P, IF-IC
Reactivity:
H Mk
Sensitivity:
Endogenous
MW (kDa):
450
Source/Isotype:
Mouse IgG1
UniProt ID:
#P78527
Entrez-Gene Id:
5591
Product Usage Information
| Application | Dilution |
|---|---|
| Western Blotting | 1:1000 |
| Immunohistochemistry (Paraffin) | 1:50 |
| Immunofluorescence (Immunocytochemistry) | 1:50 |
Storage
For a carrier free (BSA and azide free) version of this product see product #91865.
Specificity/Sensitivity
DNA-PKcs (3H6) Mouse mAb recognizes endogenous levels of total DNA-PKcs protein.
Source / Purification
Monoclonal antibody is produced by immunizing animals with a recombinant protein fragment specific to human DNA-PKcs protein expressed in E.coli.
Background
DNA-dependent protein kinase (DNA-PK) is an important factor in the repair of double-stranded breaks in DNA. Cells lacking DNA-PK or in which DNA-PK is inhibited fail to show proper nonhomologous end-joining (NHEJ) (1-7). DNA-PK is composed of two DNA-binding subunits (Ku70 and Ku86) and one 450 kDa catalytic subunit (DNA-PKcs) (8). It is thought that a heterodimer of Ku70 and Ku86 binds to double-stranded DNA broken ends before DNA-PKcs binds and is activated (1,9). Activated DNA-PKcs is a serine/threonine kinase that has been shown to phosphorylate a number of proteins in vitro, including p53, transcription factors, RNA polymerase, and Ku70/Ku86 (10,11). DNA-PKcs autophosphorylation at multiple sites, including Thr2609 and Ser2056, results in an inactivation of DNA-PK kinase activity and NHEJ ability (12,13). It has been demonstrated, however, that DNA-PK preferentially phosphorylates substrates before it autophosphorylates, suggesting that DNA-PK autophosphorylation may play a role in disassembly of the DNA repair machinery (14,15). Autophosphorylation at Thr2609 has also been shown to be required for DNA-PK-mediated double-strand break repair, and phosphorylated DNA-PK co-localizes with H2A.X and 53BP1 at sites of DNA damage (16). Phosphorylation at Ser2056 occurs in response to double-stranded DNA breaks and ATM activation (17).
Background References
- Gottlieb, T.M. and Jackson, S.P. (1993) Cell 72, 131-42.
- Hartley, K.O. et al. (1995) Cell 82, 849-56.
- Rosenzweig, K.E. et al. (1997) Clin Cancer Res 3, 1149-56.
- Jackson, S.P. and Jeggo, P.A. (1995) Trends Biochem Sci 20, 412-5.
- Roth, D.B. et al. (1995) Curr Biol 5, 496-9.
- Baumann, P. and West, S.C. (1998) Proc Natl Acad Sci U S A 95, 14066-70.
- Chen, S. et al. (2001) J Biol Chem 276, 24323-30.
- Jeggo, P.A. (1997) Mutat Res 384, 1-14.
- Suwa, A. et al. (1994) Proc Natl Acad Sci U S A 91, 6904-8.
- Anderson, C.W. and Lees-Miller, S.P. (1992) Crit Rev Eukaryot Gene Expr 2, 283-314.
- Kuhn, A. et al. (1995) Genes Dev 9, 193-203.
- Chan, D.W. and Lees-Miller, S.P. (1996) J Biol Chem 271, 8936-41.
- Douglas, P. et al. (2002) Biochem. J. 368, 243-51.
- Lees-Miller, S.P. et al. (1992) Mol Cell Biol 12, 5041-9.
- Jackson, S.P. et al. (1990) Cell 63, 155-65.
- Chan, D.W. et al. (2002) Genes Dev 16, 2333-8.
- Yajima, H. et al. (2009) J Mol Biol 385, 800-10.
Species Reactivity
Species reactivity is determined by testing in at least one approved application (e.g., western blot).
Western Blot Buffer
IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
Applications Key
W: Western Blotting IHC-P: Immunohistochemistry (Paraffin) IF-IC: Immunofluorescence (Immunocytochemistry)
Cross-Reactivity Key
H: Human M: Mouse R: Rat Hm: Hamster Mk: Monkey Vir: Virus Mi: Mink C: Chicken Dm: D. melanogaster X: Xenopus Z: Zebrafish B: Bovine Dg: Dog Pg: Pig Sc: S. cerevisiae Ce: C. elegans Hr: Horse GP: Guinea Pig Rab: Rabbit G: Goat All: All Species Expected
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