Cell Signaling Technology

Product Pathways - Chromatin Regulation / Epigenetics

Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb #8240

No. Size Price
8240S 100 µl ( 20 western blots ) ¥4,050.00 现货查询 购买询价
8240T 20 µl ( 2 western blots ) ¥1,600.00 现货查询 购买询价
8240 carrier free & custom formulation / quantityemail request
Applications Dilution Species-Reactivity Sensitivity MW (kDa) Isotype
W 1:2000 Human,Mouse,Rat,Monkey, Endogenous 23 Rabbit IgG
IP 1:100
F 1:1600
IF-IC 1:1600
ChIP 1:100

Species cross-reactivity is determined by western blot.

Applications Key: W=Western Blotting, IP=Immunoprecipitation, F=Flow Cytometry, IF-IC=Immunofluorescence (Immunocytochemistry), ChIP=Chromatin IP,

Specificity / Sensitivity

Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb recognizes endogenous levels of histone H2A protein only when ubiquitinated at Lys119. The antibody does not cross-react with other ubiquitinated proteins or free ubiquitin.

Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb兔单抗能够检测仅在Lys119位点泛素化的内源性histone H2A的蛋白水平。该抗体不能与其它泛素化的蛋白或游离的泛素发生交叉反应。

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human histone H2A protein in which Lys119 is mono-ubiquitinated.

通过合成的在Lys119单泛素化的人源histone H2A蛋白羧基端周围相应的多肽片段去免疫动物从而制备出单克隆抗体。

Chromatin IP

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 NCCIT cells and either 10 µl of Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb or 2 µl of Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using human HoxA1 intron 1 primers, SimpleChIP® Human HoxA2 Promoter Primers #5517, SimpleChIP® Human GAPDH Exon 1 Primers #5516, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin (equivalent to one).

使用SimpleChIP®Enzymatic Chromatin IP Kit (Magnetic Beads) #9003,来自4 x 106 NCCIT细胞的交联染色质以及10 µl Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb或2 µl Normal Rabbit IgG #2729进行染色质免疫沉淀实验。使用human HoxA1 intron 1 primers、SimpleChIP® Human HoxA2 Promoter Primers #5517、SimpleChIP® Human GAPDH Exon 1 Primers #5516和SimpleChIP® Human α Satellite Repeat Primers #4486,浓缩的DNA通过real-time PCR定量。在每个样品中免疫沉淀DNA的数量被当做一个相对于总input chromatin的数量的信号(这相当于一)。

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb.

使用Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb兔单抗,免疫印迹(Western blot)分析不同细胞系中Histone H2A的蛋白水平。

IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells using Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb (green) and COX IV (4D11-B3-E8) Mouse mAb #11967 (red).

Background

The nucleosome, made up of four core histone proteins (H2A, H2B, H3, and H4), is the primary building block of chromatin. Originally thought to function as a static scaffold for DNA packaging, histones have now been shown to be dynamic proteins, undergoing multiple types of post-translational modifications, including acetylation, phosphorylation, methylation, and ubiquitination (1). Ubiquitin is a conserved 76 amino acid peptide unit that can be covalently linked to many cellular proteins by the ubiquitination process. Three components are involved in this protein-ubiquitin conjugation process. Ubiquitin is first activated by forming a thioester complex with the activation component E1; the activated ubiquitin is subsequently transferred to the ubiquitin-carrier protein E2, then from E2 to ubiquitin ligase E3 for final delivery to the epsilon-NH2 of the target protein lysine residue (2). Histone H2A is mono-ubiquitinated at Lys119 by the Polycomb Repressor Complex 1 (PRC1) and is critical for transcriptional silencing of the developmental HOX genes and X chromosome inactivation (3-6). PRC1 is composed of Bmi1 and RING1A (also RING1 or RNF1), both of which act to enhance the E3 ubiquitin ligase activity of the catalytic subunit RING1B (also RING2 or RNF2) (3,4). Histone H2A is also mono-ubiquitinated at Lys119 at sites of DNA damage. This mono-ubiquitination event requires the PRC1 components Bmi1 and RING1B, in addition to another E3 ubiquitin ligase RNF8, and contributes to subsequent recruitment of the BRCA1 complex, via binding of RAP80/UIMC1 (ubiquitin interactive motif containing 1 protein) (7-10).

核小体是由四种中心组蛋白(H2A、H2B、H3和H4)组成,它是染色质的主要构件模块。起初被认为是一个DNA包装的静态支架,目前组蛋白已经被认为是一个动态蛋白,其经历多种形式的翻译后修饰,包括乙酰化作用、磷酸化作用、甲基化作用和泛素化作用(1)。泛素是一个保守的76个氨基酸多肽单位,它通过泛素化过程共价连接许多细胞内蛋白。三个成分涉及蛋白质泛素化连接过程。泛素首先通过形成一个与活化元件E1一起的硫酯复合物被激活;随后活化的泛素转移到泛素载体蛋白E2,然后从E2传递到泛素连接酶E3的靶蛋白赖氨酸残基epsilon-NH2上(2)。Histone H2A通过Polycomb Repressor Complex 1 (PRC1)使Lys119位点单泛素化,并且对于发育的HOX基因的转录沉默和X染色体的失活起着关键作用(3-6)。PRC1是由Bmi1和RING1A (也称为RING1或RNF1)组成,两者都能提高催化亚单位RING1B (也称为RING2或RNF2)的E3泛素连接酶活性(3,4)。在DNA损伤的位点Histone H2A也使Lys119位点单泛素化。除了另外E3泛素连接酶RNF8之外,这个单泛素化过程需要PRC1成分Bmi1和RING1B,并且有助于随后的BRCA1复合物的招募,这主要通过RAP80/UIMC1(ubiquitin interactive motif containing 1 protein)的结合(7-10)。

  1. Peterson, C.L. and Laniel, M.A. (2004) Curr Biol 14, R546-51.
  2. Liu, F. and Walters, K.J. (2010) Trends Biochem Sci 35, 352-60.
  3. Wang, H. et al. (2004) Nature 431, 873-8.
  4. Cao, R. et al. (2005) Mol Cell 20, 845-54.
  5. de Napoles, M. et al. (2004) Dev Cell 7, 663-76.
  6. Fang, J. et al. (2004) J Biol Chem 279, 52812-5.
  7. Ginjala, V. et al. (2011) Mol Cell Biol 31, 1972-82.
  8. Bergink, S. et al. (2006) Genes Dev 20, 1343-52.
  9. Marteijn, J.A. et al. (2009) J Cell Biol 186, 835-47.
  10. Wu, J. et al. (2009) Mol Cell Biol 29, 849-60.

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