Cell Signaling Technology

Product Pathways - Chromatin Regulation / Epigenetics

Acetyl-Histone H2B (Lys5) (D5H1S) XP® Rabbit mAb #12799

No. Size Price
12799S 100 µl ( 10 western blots ) ¥4,050.00 现货查询 购买询价
12799T 20 µl ( 2 western blots ) ¥1,600.00 现货查询 购买询价
12799 carrier free & custom formulation / quantityemail request
Applications Dilution Species-Reactivity Sensitivity MW (kDa) Isotype
W 1:1000 Human,Mouse,Rat,Monkey, Endogenous 14 Rabbit IgG
IP 1:100
IHC-P 1:1000
IF-IC 1:400
ChIP 1:50

Species cross-reactivity is determined by western blot.

Applications Key: W=Western Blotting, IP=Immunoprecipitation, IHC-P=Immunohistochemistry (Paraffin), IF-IC=Immunofluorescence (Immunocytochemistry), ChIP=Chromatin IP,

Homology

Species predicted to react based on 100% sequence homology: Hamster, Chicken, Zebrafish, Bovine, Horse,

Specificity / Sensitivity

Acetyl-Histone H2B (Lys5) (D5H1S) XP® Rabbit mAb recognizes endogenous levels of histone H2B only when acetylated at Lys5. This antibody does not cross-react with other acetylated histones.

Acetyl-Histone H2B (Lys5) (D5H1S) XP® Rabbit mAb 兔单抗能够检测仅在Lys5位点乙酰化的内源性histone H2B蛋白水平。该抗体不能与其它乙酰化的组蛋白发生交叉反应。

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding acetylated Lys5 of human histone H2B protein.

该单克隆抗体是采用合成的与人源histone H2B蛋白Lys5位点周围相应的乙酰化片段免疫动物而制备的。

IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells, untreated (right) or treated with Trichostatin A (TSA) #9950 (left), using Acetyl-Histone H2B (Lys5) (D5H1S) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).采用共聚焦免疫荧光术检测untreated (左图)或treated with TSA #9950 (右图)的Hela细胞,使用的抗体为Acetyl-Histone H2B (Lys5) (D5H1S) XP®Rabbit mAb (绿色)。肌动蛋白微丝使用DY-554 鬼笔环肽进行标记(红色),假蓝色= DRAQ5® #4084 (DNA荧光染料)。

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Acetyl-Histone H2B (Lys5) (D5H1S) XP® Rabbit mAb in the presence of non-acetyl peptide (left) or Lys5 acetyl peptide (right).免疫组化检测石蜡包埋的人结肠癌,所使用的抗体为Acetyl-Histone H2B (Lys5) (D5H1S) XP® Rabbit mAb,左图无乙酰化肽段存在,右图有Lys5位点乙酰化肽段存在。

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Acetyl-Histone H2B (Lys5) (D5H1S) XP® Rabbit mAb.免疫组化检测石蜡包埋的人乳腺癌,所使用的抗体为Acetyl-Histone H2B (Lys5) (D5H1S) XP® Rabbit mAb。

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa and C6 cells, untreated (-) or treated with Trichostatin A (TSA) #9950 (1 μM, 18hr; +), using Acetyl-Histone H2B (Lys5) (D5H1S) XP® Rabbit mAb (upper) or Histone H2B (D2H6) Rabbit mAb #12364 (lower).Western blot检测untreated (-)或 treated with Trichostatin A (TSA) #9950 (1 μM, 18hr; +)的HeLa和C6细胞,使用的抗体为Acetyl-Histone H2B (Lys5) (D5H1S) XP®Rabbit mAb (上图) or Histone H2B (D2H6) Rabbit mAb #12364 (下图)。

IP

IP

Immunoprecipitation of acetylated histone H2B from HeLa cell extracts treated with Trichostatin A (TSA) #9950 (1 μM, 18hr) using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Acetyl-Histone H2B (D5H1S) XP® Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Acetyl-Histone H2B (D5H1S) XP® Rabbit mAb.从 经Trichostatin A (TSA) #9950 (1 μM, 18hr) 处理的HeLa细胞提取物中免疫沉淀乙酰化的histone H2B,使用的抗体为Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) 或 Acetyl-Histone H2B (D5H1S) XP® Rabbit mAb (lane 3)。Lane 1为10% input。 使用Acetyl-Histone H2B (D5H1S) XP® Rabbit mAb进行Western blot检测。

ELISA-Peptide

ELISA-Peptide

Acetyl-Histone H2B (Lys5) (D5H1S) XP® Rabbit mAb specificity was determined by peptide ELISA. The graph depicts the binding of the antibody to pre-coated acetyl-histone H2B (Lys5) peptide in the presence of increasing concentrations of various competitor peptides. As shown, only the acetyl-histone H2B (Lys5) peptide competed away binding of the antibody.Peptide ELISA方法测定Acetyl-Histone H2B (Lys5) (D5H1S) XP® Rabbit mAb 抗体特异性。 图片显示在不断提高不同竞争性多肽的浓度时抗体都能够与预包被好的acetyl-histone H2B (Lys5) peptide结合。同时也显示了,只有acetyl-histone H2B (Lys5) peptide能够竞争性抑制其与抗体的结合。

Chromatin IP

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HeLa cells and either 10 µl of Acetyl-Histone H2B (Lys5) (D5H1S) 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 SimpleChIP® Human GAPDH Exon 1 Primers #5516, SimpleChIP® Human RPL30 Exon 3 Primers #7014, SimpleChIP® Human MyoD1 Exon 1 Primers #4490, 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, which is equivalent to one.使用SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003 对4 x 106 HeLa细胞中交联过的染色质进行染色质免疫沉淀,所用抗体为10 µl of Acetyl-Histone H2B (Lys5) (D5H1S) XP® Rabbit mAb 或2 µl Normal Rabbit IgG #2729,富集到的DNA经过实时PCR定量,所使用产品为SimpleChIP® Human GAPDH Exon 1 Primers #5516, SimpleChIP®Human RPL30 Exon 3 Primers #7014, SimpleChIP® Human MyoD1 Exon 1 Primers #4490, 以及SimpleChIP® Human α Satellite Repeat Primers #4486。每个样品中免疫沉淀得到的DNA量由与input chromatin( 相当于1)的相对信号值来表示。

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 (1,2). The p300/CBP histone acetyltransferases acetylate multiple lysine residues in the amino terminal tail of histone H2B (Lys5, 12, 15, and 20) at gene promoters during transcriptional activation (1-3). Hyper-acetylation of the histone tails neutralizes the positive charge of these domains and is believed to weaken histone-DNA and nucleosome-nucleosome interactions, thereby destabilizing chromatin structure and increasing the access of DNA to various DNA-binding proteins (4,5). In addition, acetylation of specific lysine residues creates docking sites that facilitate recruitment of many transcription and chromatin regulatory proteins that contain a bromodomain, which binds to acetylated lysine residues (6). Histone H2B is mono-ubiquitinated at Lys120 during transcriptional activation by the RAD6 E2 protein in conjunction with the BRE1A/BRE1B E3 ligase (also known as RNF20/RNF40) (7). Mono-ubiquitinated histone H2B Lys120 is associated with the transcribed region of active genes and stimulates transcriptional elongation by facilitating FACT-dependent chromatin remodeling (7-9). In addition, it is essential for subsequent methylation of histone H3 Lys4 and Lys79, two additional histone modifications that regulate transcriptional initiation and elongation (10). In response to metabolic stress, AMPK is recruited to responsive genes and phosphorylates histone H2B at Lys36, both at promoters and in transcribed regions of genes, and may regulate transcriptional elongation (11). In response to multiple apoptotic stimuli, histone H2B is phosphorylated at Ser14 by the Mst1 kinase (12). Upon induction of apoptosis, Mst1 is cleaved and activated by caspase-3, leading to global phosphorylation of histone H2B during chromatin condensation. Interestingly, histone H2B is rapidly phosphorylated at irradiation-induced DNA damage foci in mouse embryonic fibroblasts (13). In this case, phosphorylation at Ser14 is rapid, depends on prior phosphorylation of H2AX Ser139, and occurs in the absence of apoptosis, suggesting that Ser14 phosphorylation may have distinct roles in DNA-damage repair and apoptosis.

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(1,2)。在转录激活期间,p300/CBP组蛋白乙酰化转移酶在histone H2B (Lys5、12、15和20)基因启动子的氨基端使多个赖氨酸残基乙酰化 (1-3)。组蛋白尾部的高度乙酰化中和了这些区域的正电荷,并且被认为弱化了组蛋白-DNA和核小体-核小体的相互作用,因此使核染色质不稳定以及增加了DNA与不同的DNA结合蛋白的接近程度(4,5)。此外,特定的赖氨酸残基乙酰化产生了停留点,这些位置有助于招募许多转录和染色质调节蛋白,这些蛋白包含一个bromodomain,该区域能结合到乙酰化的赖氨酸残基(6)。在转录激活期间,通过RAD6 E2蛋白连接BRE1A/BRE1B E3 ligase (also known as RNF20/RNF40)从而使Histone H2B在Lys120位点发生单泛素化(7)。单泛素化的histone H2B Lys120是与活化基因的转录区域有关联,并且通过促进FACT依赖的染色质重构从而刺激转录延伸(7-9)。此外,对于随后的histone H3在Lys4 和Lys79位点的甲基化有本质作用,这两个附加的组蛋白修饰能调节转录起始和延伸(10)。在代谢应激的反应下,AMPK被招募到反应性基因以及使histone H2B在Lys36位点磷酸化,这都发生在基因的启动子和转录区域,以及这些可能调节转录的延伸(11)。在多种凋亡刺激下,histone H2B通过Mst1激酶使其Ser14位点磷酸化(12)。在凋亡的诱导下,Mst1激酶被剪切,并且通过caspase-3激活,导致在染色质浓缩期间histone H2B整体的磷酸化。有趣的是,在小鼠胚胎成纤维细胞中histone H2B在照射诱导DNA损伤点发生快速磷酸化(13)。既然这样,在Ser14位点的磷酸化是快速的,这取决于在H2AX Ser139位点的优先磷酸化,并且是发生在没有凋亡情况下,从而认为Ser14位点磷酸化可能在DNA损伤的修复和凋亡中有明显的作用。

  1. Peterson, C.L. and Laniel, M.A. (2004) Curr Biol 14, R546-51.
  2. Jaskelioff, M. and Peterson, C.L. (2003) Nat Cell Biol 5, 395-9.
  3. Roth, S.Y. et al. (2001) Annu Rev Biochem 70, 81-120.
  4. Workman, J.L. and Kingston, R.E. (1998) Annu Rev Biochem 67, 545-79.
  5. Hansen, J.C. et al. (1998) Biochemistry 37, 17637-41.
  6. Yang, X.J. (2004) Bioessays 26, 1076-87.
  7. Kim, J. et al. (2009) Cell 137, 459-71.
  8. Minsky, N. et al. (2008) Nat Cell Biol 10, 483-8.
  9. Pavri, R. et al. (2006) Cell 125, 703-17.
  10. Shilatifard, A. (2006) Annu Rev Biochem 75, 243-69.
  11. Bungard, D. et al. (2010) Science 329, 1201-5.
  12. Cheung, W.L. et al. (2003) Cell 113, 507-17.
  13. Fernandez-Capetillo, O. et al. (2004) J Exp Med 199, 1671-7.

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

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

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

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