Revision 3
#9720
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For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:
FC-FP
Reactivity:
H M R Mk
Sensitivity:
Endogenous
Source/Isotype:
Rabbit IgG
UniProt ID:
#P16104
Entrez-Gene Id:
3014
Product Usage Information
Application | Dilution |
---|---|
Flow Cytometry (Fixed/Permeabilized) | 1:50 |
Storage
Specificity/Sensitivity
Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb detects endogenous levels of H2A.X only when phosphorylated at serine 139.
Source / Purification
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser139 of human H2A.X. The antibody was conjugated to Alexa Fluor® 647 under optimal conditions with an F/P ratio of 2-5. The Alexa Fluor® 647 dye is maximally excited by red light (e.g. 633 nm He-Ne laser). Antibody conjugates of the Alexa Fluor® 647 dye produce bright far-red-fluorescence emission, with a peak at 665 nm.
Description
This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 647 fluorescent dye and tested in-house for direct flow cytometry and immunofluorescent analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb #9718.
Background
Histone H2A.X is a variant histone that represents approximately 10% of the total H2A histone proteins in normal human fibroblasts (1). H2A.X is required for checkpoint-mediated cell cycle arrest and DNA repair following double-stranded DNA breaks (1). DNA damage, caused by ionizing radiation, UV-light, or radiomimetic agents, results in rapid phosphorylation of H2A.X at Ser139 by PI3K-like kinases, including ATM, ATR, and DNA-PK (2,3). Within minutes following DNA damage, H2A.X is phosphorylated at Ser139 at sites of DNA damage to generate γ-H2A.X (4). This very early event in the DNA-damage response is required for recruitment of a multitude of DNA-damage response proteins, including MDC1, NBS1, RAD50, MRE11, 53BP1, and BRCA1 (1). In addition to its role in DNA-damage repair, H2A.X is required for DNA fragmentation during apoptosis and is phosphorylated by various kinases in response to apoptotic signals. H2A.X is phosphorylated at Ser139 by DNA-PK in response to cell death receptor activation, c-Jun N-terminal Kinase (JNK1) in response to UV-A irradiation, and p38 MAPK in response to serum starvation (5-8). H2A.X is constitutively phosphorylated on Tyr142 in undamaged cells by WSTF (Williams-Beuren syndrome transcription factor) (9,10). Upon DNA damage, and concurrent with phosphorylation of Ser139, Tyr142 is dephosphorylated at sites of DNA damage by recruited EYA1 and EYA3 phosphatases (9). While phosphorylation at Ser139 facilitates the recruitment of DNA repair proteins and apoptotic proteins to sites of DNA damage, phosphorylation at Tyr142 appears to determine which set of proteins are recruited. Phosphorylation of H2A.X at Tyr142 inhibits the recruitment of DNA repair proteins and promotes binding of pro-apoptotic factors such as JNK1 (9). Mouse embryonic fibroblasts expressing only mutant H2A.X Y142F, which favors recruitment of DNA repair proteins over apoptotic proteins, show a reduced apoptotic response to ionizing radiation (9). Thus, it appears that the balance of H2A.X Tyr142 phosphorylation and dephosphorylation provides a switch mechanism to determine cell fate after DNA damage.
Background References
- Yuan, J. et al. (2010) FEBS Lett 584, 3717-24.
- Rogakou, E.P. et al. (1998) J Biol Chem 273, 5858-68.
- Burma, S. et al. (2001) J Biol Chem 276, 42462-7.
- Rogakou, E.P. et al. (1999) J Cell Biol 146, 905-16.
- Mukherjee, B. et al. (2006) DNA Repair (Amst) 5, 575-90.
- Solier, S. et al. (2009) Mol Cell Biol 29, 68-82.
- Lu, C. et al. (2006) Mol Cell 23, 121-32.
- Lu, C. et al. (2008) FEBS Lett 582, 2703-8.
- Cook, P.J. et al. (2009) Nature 458, 591-6.
- Xiao, A. et al. (2009) Nature 457, 57-62.
Species Reactivity
Species reactivity is determined by testing in at least one approved application (e.g., western blot).
Applications Key
FC-FP: Flow Cytometry (Fixed/Permeabilized)
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
Trademarks and Patents
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
This product is provided under an intellectual property license from Life Technologies Corporation. The transfer of this product is conditioned on the buyer using the purchased product solely in research conducted by the buyer, excluding contract research or any fee for service research, and the buyer must not (1) use this product or its components for (a) diagnostic, therapeutic or prophylactic purposes; (b) testing, analysis or screening services, or information in return for compensation on a per-test basis; or (c) manufacturing or quality assurance or quality control, and/or (2) sell or transfer this product or its components for resale, whether or not resold for use in research. For information on purchasing a license to this product for purposes other than as described above, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or [email protected].
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.
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