Cell Signaling Technology

Product Pathways - Chromatin Regulation / Epigenetics

Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (Alexa Fluor® 488 Conjugate) #9719

Applications Dilution Species-Reactivity Sensitivity MW (kDa) Isotype
F 1:50 Human,Mouse,Rat,Monkey, Endogenous Rabbit IgG

Species cross-reactivity is determined by western blot.

Applications Key: F=Flow Cytometry,

Specificity / Sensitivity

Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb detects endogenous levels of H2A.X only when phosphorylated at serine 139.

Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb兔单抗能够检测仅在serine 139位点磷酸化的内源性H2A.X的总蛋白水平。

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® 488 under optimal conditions with an F/P ratio of 2-5.

通过合成的与人源H2A.X蛋白Ser139位点周围相应的磷酸化片段去免疫动物从而制备出此单克隆抗体。该抗体以F/P比率2-5的最佳条件偶联了Alexa Fluor® 488。


This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 488 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.

Cell Signaling Technology antibody连接着Alexa Fluor® 488 fluorescent dye,并且用直标流式细胞检测和免疫荧光来分析方法检测人类细胞。该抗体可能与非偶联的Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb #9718有同一物种的交叉反应。

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® 488 Conjugate).

使用Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb兔单抗 (Alexa Fluor® 488 Conjugate)标记,流式细胞仪分Jurkat细胞,细胞分为untreated (绿色)或etoposide-treated (蓝色)。



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® 488 Conjugate) (green) and beta-Tubulin Antibody #2146 (red).

使用Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb 兔单抗(Alexa Fluor® 488 Conjugate) (绿色)和beta-Tubulin Antibody #2146 (红色)双标记,共聚焦免疫荧光分析NIH/3T3细胞,细胞分为untreated (左图)或etoposide-treated (右图)。


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.

在正常人源成纤维细胞中,组蛋白H2A.X是一种变异的组蛋白,大约占H2A组蛋白总量的10%(1)。在双链DNA中断之后,checkpoint介导的细胞周期终止和DNA修复都需要H2A.X的参与(1)。由电离辐射、紫外线或辐射物质引起的DNA损伤导致由PI3K-like激酶包括ATM、ATR和DNA-PK催化H2A.X蛋白在Ser13位点的快速磷酸化(2,3)。在DNA损伤后的数分钟内,H2A.X将会在DNA损伤部位的Ser139位点上发生磷酸化(4)。在DNA的损伤反应下,这个非常早期事件对于多个DNA损伤反应蛋白包括MDC1、NBS1、RAD50、MRE11、53BP1和BRCA1的招募是需要的(1)。除了在DNA的损伤修复中起作用,在细胞凋亡期间,对于DNA的片段化是需要H2A.X,并且在凋亡信号刺激下不同的激酶被磷酸化。通过在死亡受体的活化下的DNA-PK、在UV-A辐射下的c-Jun N-terminal Kinase (JNK1)以及在血清饥饿下的p38 MAPK ,促使H2A.X在Ser139位点磷酸化(5-8)。在受损的细胞中,通过WSTF(Williams-Beuren syndrome transcription factor)促使H2A.X在Tyr142位点持续地磷酸化(9,10)。在DNA损伤发生时,通过招募EYA1和EYA3磷酸酶使在DNA损伤位点已磷酸化的Ser139、Tyr142位点发生去磷酸化(9)。当在Ser139位点磷酸化有利于招募DNA修复蛋白和凋亡蛋白到损伤部位的时候,Tyr142位点的磷酸化似乎确定这类蛋白被招募。H2A.X在Tyr142位点的磷酸化将阻止DNA修复蛋白的募集,并且促进凋亡前因子例如JNK1的结合(9)。在只表达突变H2A,X(Y142F)的鼠胚胎成纤维细胞中,该细胞相对于凋亡蛋白更倾向于DNA修复蛋白的招募,其在电离辐射反应下显示有个降低的凋亡反应(9)。因此,H2A.X的Tyr142位点磷酸化和去磷酸化之间的平衡提供了一个在DNA受损后决定细胞命运的开关机制。

  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

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

U.S. Patent No. 5,675,063.

The Alexa Fluor dye antibody conjugates in this product are sold under license from Life Technologies Corporation for research use only, except for use in combination with DNA microarrays. The Alexa Fluor® dyes (except for Alexa Fluor® 430 dye) are covered by pending and issued patents. Alexa Fluor® is a registered trademark of Molecular Probes, Inc.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.

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