| 货号 | 8173S |
| 反应种属 | Human/Mouse/Rat/Monkey |
| 来源宿主 | Rabbit |
| 应用 | W/IF-IC/ChIP |
| 目标/特异性 | Acetyl-Histone H3 (Lys27) (D5E4) XP® Rabbit mAb recognizes endogenous levels of histone H3 protein only when acetylated at Lys27. This antibody does not cross react with histone H3 acetylated at Lys9, 14, 18, 23, or 56. |
| 使用方法 | WB(1:1000) IF-IC (1:200) ChIP (1:100) |
| 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). Histone acetylation occurs mainly on the amino-terminal tail domains of histones H2A (Lys5), H2B (Lys5, 12, 15, and 20), H3 (Lys9, 14, 18, 23, 27, and 56), and H4 (Lys5, 8, 12, and 16) and is important for the regulation of histone deposition, transcriptional activation, DNA replication, recombination, and DNA repair (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 accessibility of DNA to various DNA-binding proteins (4,5). In addition, acetylation of specific lysine residues creates docking sites for a protein module called the bromodomain, which binds to acetylated lysine residues (6). Many transcription and chromatin regulatory proteins contain bromodomains and may be recruited to gene promoters, in part, through binding of acetylated histone tails. Histone acetylation is mediated by histone acetyltransferases (HATs), such as CBP/p300, GCN5L2, PCAF, and Tip60, which are recruited to genes by DNA-bound protein factors to facilitate transcriptional activation (3). Deacetylation, which is mediated by histone deacetylases (HDAC and sirtuin proteins), reverses the effects of acetylation and generally facilitates transcriptional repression (7,8). |
| 存放说明 | -20C |
| 计算分子量 | 17 |
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 H3 (Lys27) (D5E4) XP® Rabbit mAb (upper) or Histone H3 (D1H2) XP® Rabbit mAb #4499 (lower). 使用Acetyl-Histone H3 (Lys27) (D5E4) XP® Rabbit mAb 兔单抗(上图)和Histone H3 (D1H2) XP® Rabbit mAb 兔单抗#4499 (下图)，免疫印迹(Western blot)分析HeLa和C2C12细胞中Acetyl-Histone H3 (Lys27)和Histone H3蛋白水平，细胞分为untreated或treated (+) with Trichostatin A (TSA) #9950 (1 μM, 18 hr)。 | |
Acetyl-Histone H3 (Lys27) (D5E4) XP® Rabbit mAb specificity was determined by peptide ELISA. The graph depicts the binding of the antibody to precoated acetyl-histone H3 (Lys27) peptide in the presence of increasing concentrations of various competitor peptides. As shown, only the acetyl-histone H3 (Lys27) peptide competed away binding of the antibody. 通过peptide ELISA确定Acetyl-Histone H3 (Lys27) (D5E4) 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 H3 (Lys27) peptide的结合能力，并且多肽中含有不断浓度增加的不同竞争多肽。如同所示，仅acetyl-histone H3 (Lys27) peptide竞争脱离抗体的结合。 | |
Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HeLa cells and either 5 μl of Acetyl-Histone H3 (Lys27) (D5E4) 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 AFM Intron 1 Primers #5098, 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，来自4 x 106 HeLa细胞的交联染色质以及5 µl Acetyl-Histone H3 (Lys27) (D5E4) 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 AFM Intron 1 Primers #5098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。 | |
Confocal immunofluorescent analysis of HeLa cells, untreated (left) or treated with Trichostatin A (TSA) #9950 (1 uM, 4 hr; right), using Acetyl-Histone H3 (Lys27) (D5E4) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). 使用Acetyl-Histone H3 (Lys27) (D5E4) XP® 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)或处理Trichostatin A (TSA) #9950 (1 uM, 4 hr; 右图)。DY-554 phalloidin标记微丝蛋白(红色)。 |
