Cell Signaling Technology

Product Pathways - Chromatin Regulation / Epigenetics

Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb #9718

h2ax  

No. Size Price
9718S 100 µl ( 10 western blots ) ¥3,780.00 现货查询 购买询价
9718T 20 µl ( 2 western blots ) ¥1,500.00 现货查询 购买询价
9718 carrier free & custom formulation / quantityemail request
Applications Dilution Species-Reactivity Sensitivity MW (kDa) Isotype
W 1:1000 Human,Mouse,Rat,Monkey, Endogenous 15 Rabbit IgG
IHC-P 1:480
F 1:200
IF-IC 1:200

Species cross-reactivity is determined by western blot.

Applications Key: W=Western Blotting, IHC-P=Immunohistochemistry (Paraffin), F=Flow Cytometry, IF-IC=Immunofluorescence (Immunocytochemistry),

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能够检测仅在serine139位点磷酸化的内源性H2A.X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.

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。

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human lung carcinoma untreated (left) or lambda-phosphatase-treated (right), using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb.

使用Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb兔单抗,免疫组化分析人源肺癌组织石蜡切片,左图是untreated,右图是lambda-phosphatase-treated。

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb in the presence of control peptide (left) or Phospho-Histone H2A.X (Ser139) Blocking Peptide #1260 (right).

使用Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb兔单抗,左图是control peptide,右图是Phospho-Histone H2A.X (Ser139) Blocking Peptide #1260,免疫组化分析人源乳腺癌组织石蜡切片。

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of HeLa cells, untreated (blue) or UV-treated (green), using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb compared to a nonspecific negative control antibody (red).

与一个非特异阴性control antibody (红色)比较,使用Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb进行流式细胞仪分析HeLa细胞,细胞分为untreated (蓝色)或UV-treated (绿色)。

Western Blotting

Western Blotting

Western blot analysis of extracts from untreated or UV-treated 293 cells, using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (upper) or Histone H2A.X Antibody #2595 (lower).

使用Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb兔单抗 (上图)或Histone H2A.X Antibody #2595 (下图),免疫印迹(Western blot)分析untreated或UV-treated 293细胞系中Phospho-Histone H2A.X和Histone H2A.X的蛋白水平。

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb, showing nuclear localization.

使用Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb兔单抗,免疫组化分析人源肺癌石蜡切片,结果显示为细胞核定位。

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human colon carcinoma, using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb.

使用Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb兔单抗,免疫组化分析人源结肠癌石蜡切片。

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded HT-29 cells untreated (left) or UV-treated (right), using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb.

使用Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb兔单抗,免疫组化分析HT-29细胞石蜡切片,细胞分为untreated (左图)或UV-treated (右图)。

IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells, untreated (left) or UV-treated (right), using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).

使用Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb 兔单抗(绿色),共聚焦免疫荧光分析HeLa细胞,细胞分为untreated (左图)或UV-treated (右图)。DY-554 phalloidin标记微丝蛋白(红色)。

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.

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.

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

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

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