| 货号 | 9083S |
| 反应种属 | Human/Mouse/Rat/Monkey |
| 来源宿主 | Rabbit |
| 应用 | W/IP/IF-IC/ChIP |
| 目标/特异性 | Acetyl-Histone H2B (Lys15) (D8H1) XP® Rabbit mAb recognizes endogenous levels of histone H2B protein only when acetylated at Lys15. This antibody does not cross-react with histone H2B acetylated at Lys5, Lys12, or Lys20. |
| 使用方法 | WB(1:1000) IP (1:50) IF-IC (1:1600) ChIP (1:50) |
| 供应商 | CST |
| 背景 | 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. |
| 存放说明 | -20C |
| 计算分子量 | 14 |
Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HeLa cells and either 10 µl of Acetyl-Histone H2B (Lys15) (D8H1) 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，用4 x 106 HeLa细胞的交联染色质以及10 µl Acetyl-Histone H2B (Lys15) (D8H1) XP® Rabbit mAb或2 µl Normal Rabbit IgG #2729进行染色质免疫沉淀实验。使用SimpleChIP® Human GAPDH Exon 1 Primers #5516、SimpleChIP® Human RPL30 Exon 3 Primers #7014、SimpleChIP® Human MyoD1 Exon 1 Primers #4490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1的总input chromatin的数量的信号。 | |
Confocal immunofluorescent analysis of HeLa cells, untreated (left) or treated with TSA #9950 (right) using Acetyl-Histone H2B (Lys15) (D8H1) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye). 使用Acetyl-Histone H2B (Lys15) (D8H1) XP® Rabbit mAb 兔单抗(绿色)标记，共聚焦免疫荧光分析HeLa细胞，细胞分为没处理 (左图)或 TSA处理 #9950 (右图)。DY-554 phalloidin标记微丝蛋白(红色)。蓝色= DRAQ5® #4084 (DNA荧光染料)。 | |
Western blot analysis of extracts from HeLa and C2C12 cells, untreated (-) or treated (+) with Trichostatin A (TSA) #9950 (1 μM, 18 hr), using Acetyl-Histone H2B (Lys15) (D8H1) XP® Rabbit mAb (upper) and Histone H2B (V119) Antibody #8135 (lower). 使用Acetyl-Histone H2B (Lys15) (D8H1) XP® Rabbit mAb兔单抗 (上图)和Histone H2B (V119) Antibody #8135 (下图)，免疫印迹(Western blot)分析HeLa和C2C12细胞中Acetyl-Histone H2B (Lys15)蛋白水平细胞分为untreated (-)或treated (+) with Trichostatin A (TSA) #9950 (1 μM, 18 hr)。 | |
Acetyl-Histone H2B (Lys15) (D8H1) XP® Rabbit mAb specificity was determined by peptide ELISA. The graph depicts the binding of the antibody to pre-coated acetyl-histone H2B (Lys15) peptide in the presence of increasing concentrations of various competitor peptides. As shown, only the acetyl-histone H2B (Lys15) peptide competed away binding of the antibody. 通过peptide ELISA确定Acetyl-Histone H2B (Lys15) (D8H1) XP® Rabbit mAb兔单抗的特异性。该图描述了抗体与提前包被的acetyl-histone H2B (Lys15) peptide的结合能力，并且多肽中含有浓度递增的不同竞争多肽。如同所示，仅acetyl-histone H2B (Lys15) peptide竞争脱离抗体的结合。 |
