Cell Signaling Technology

Product Pathways - Chromatin Regulation / Epigenetics

Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (Alexa Fluor® 647 Conjugate) #9720

Applications Reactivity Sensitivity Isotype
IF-IC F H M Endogenous Rabbit IgG

Applications Key:  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

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.

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of Jurkat cells, untreated (green) or etoposide-treated (blue), using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (Alexa Fluor® 647 Conjugate).

IF-IC

IF-IC

Confocal immunofluorescent analysis of NIH/3T3 cells, untreated (left) or etoposide-treated (right), double-labeled with Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (Alexa Fluor® 647 Conjugate) (blue) and beta-Tubulin Antibody #2146 (red).

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 (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.

  1. Yuan, J. et al. (2010) FEBS Lett 584, 3717-24.
  2. Rogakou, E.P. et al. (1998) J Biol Chem 273, 5858-68.
  3. Burma, S. et al. (2001) J Biol Chem 276, 42462-7.
  4. Rogakou, E.P. et al. (1999) J Cell Biol 146, 905-16.
  5. Mukherjee, B. et al. (2006) DNA Repair (Amst) 5, 575-90.
  6. Solier, S. et al. (2009) Mol Cell Biol 29, 68-82.
  7. Lu, C. et al. (2006) Mol Cell 23, 121-32.
  8. Lu, C. et al. (2008) FEBS Lett 582, 2703-8.
  9. Cook, P.J. et al. (2009) Nature 458, 591-6.
  10. Xiao, A. et al. (2009) Nature 457, 57-62.

Application References

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Companion Products

Rabbit Monoclonals Produced Using Epitomics® Technology, U.S. Patent No. 5,675,063.Alexa Fluor® is a registered trademark of Molecular Probes, Inc.

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

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