| 货号 | 5546T |
| 同种亚型 | Rabbit IgG |
| 反应种属 | Human,Mouse,Rat,Monkey, |
| 来源宿主 | Rabbit IgG |
| 应用 | WB, F , IF-IC , ChIP |
| 目标/特异性 | Ubiquityl-Histone H2B (Lys120) (D11) XP® Rabbit mAb detects endogenous levels of histone H2B protein only when ubiquitylated on Lys120. The antibody does not cross-react with other ubiquitylated proteins or free ubiquitin. |
| 使用方法 | WB(1:1000) F (1:100) IF-IC (1:800) ChIP (1:100) |
| 供应商 | CST |
| 灵敏度 | Endogenous |
| 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 ubiquitylation (1). Ubiquitin is a conserved 76 amino acid peptide unit that can be covalently linked to many cellular proteins by the ubiquitylation process. Three components are involved in this protein-ubiquitin conjugation process. Ubiquitin is first activated by forming a thiolester 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 H2B is mono-ubiquitylated on lysine 120 during transcriptional activation by the RAD6 E2 protein in conjunction with the BRE1A/BRE1B E3 ligase (also known as RNF20/RNF40) (3). The RAD6/BRE1 complex is recruited to gene promoters during activation by the PAF complex, an RNA polymerase II-associated protein complex that regulates transcriptional elongation (3-5). Mono-ubiquitylated histone H2B lysine 120 is associated with the transcribed region of active genes (3,6). Mono-ubiquitylation of histone H2B stimulates transcriptional elongation by facilitating FACT-dependent chromatin remodeling (7,8). In addition, it is essential for subsequent methylation of histone H3 lysines 4 and 79, two additional histone modifications that regulate transcriptional initiation and elongation (9). Interestingly, de-ubiquitylation of histone H2B lysine 120 by USP22, a subunit of the human SAGA histone acetyltransferase complex, is a required step in transcriptional activation (10). Thus, it appears that the ubiquitylation state of histone H2B is dynamic during transcription and may serve as an intermediate step in transcriptional activation. |
| 存放说明 | -20C |
| 计算分子量 | 23 |
| 参考文献 | 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 . Kim, J. et al. (2009) Cell 137, 459-71. 4 . Wood, A. et al. (2003) J Biol Chem 278, 34739-42. 5 . Xiao, T. et al. (2005) Mol Cell Biol 25, 637-51. 6 . Minsky, N. et al. (2008) Nat Cell Biol 10, 483-8. 7 . Pavri, R. et al. (2006) Cell 125, 703-17. 8 . Fleming, A.B. et al. (2008) Mol Cell 31, 57-66. 9 . Shilatifard, A. (2006) Annu Rev Biochem 75, 243-69. 10 . Wyce, A. et al. (2004) Novartis Found Symp 259, 63-73; discussion 73-7, 163-9. |
Confocal immunofluorescent analysis of HeLa cells using Ubiquityl-Histone H2B (Lys120) (D11) XP® Rabbit mAb (green) and COX IV (4D11-B3-E8) Mouse mAb #11967 (red). | |
Western blot analysis of extracts from various cell lines using Ubiquityl-Histone H2B (Lys120) (D11) XP® Rabbit mAb. 使用Ubiquityl-Histone H2B (Lys120) (D11) XP® Rabbit mAb兔单抗，免疫印迹(Western blot)分析不同细胞中Ubiquityl-Histone H2B (Lys120)的蛋白水平。 | |
Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HeLa cells and either 10 μl of Ubiquityl-Histone H2B (Lys120) (D11) 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 γ-Actin Promoter Primers #5037, SimpleChIP® Human γ-Actin Intron 3 Primers #5047, SimpleChIP® Human GAPDH Promoter Primers #4471, and SimpleChIP® Human GAPDH Intron 2 Primers #4478. 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 Ubiquityl-Histone H2B (Lys120) (D11) 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 γ-Actin Promoter Primers #5037、SimpleChIP® Human γ-Actin Intron 3 Primers #5047、SimpleChIP® Human GAPDH Promoter Primers #4471和SimpleChIP® Human GAPDH Intron 2 Primers #4478，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。 |
