| 货号 | 7228S |
| 描述 | The PathScan® Acetyl-Histone H4 (Lys12) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of histone H4 when acetylated at lysine 12. A histone H4 mouse antibody has been coated onto the microwells. After incubation with cell lysates, histone H4 protein (acetylated and non-acetylated) is captured by the coated antibody. Following extensive washing, Acetyl-histone H4 (Lys12) rabbit antibody is added to detect acetylated Lys12 on the histone H4 protein. Anti-rabbit IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of histone H4 acetylated at Lys12. Antibodies in kit are custom formulations specific to kit. |
| 反应种属 | Human/Mouse |
| 应用 | ELISA |
| 目标/特异性 | CSTs PathScan® Acetyl-Histone H4 (Lys12) Sandwich ELISA Kit #7228 detects endogenous levels of histone H4 when acetylated at Lys12. As shown in Figure 1 using the Acetyl-Histone H4 (Lys12) Sandwich ELISA Kit #7228, a high level of acetylation at Lys12 on histone H4 is detected in NIH/3T3 cells when treated with TSA. The level of total histone H4 (acetylated and non-acetylated) remains unchanged as shown by Western analysis (Figure 1). Similar results are obtained when COS and Jurkat cells are treated with TSA (data not shown). This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species. |
| 供应商 | CST |
| 背景 | Modulation of chromatin structure plays an important role in the regulation of transcription in eukaryotes. The nucleosome, made up of DNA wound around eight core histone proteins (two each of H2A, H2B, H3, and H4), is the primary building block of chromatin (1). The amino-terminal tails of core histones undergo various post-translational modifications, including acetylation, phosphorylation, methylation, and ubiquitination (2-5). These modifications occur in response to various stimuli and have a direct effect on the accessibility of chromatin to transcription factors and, therefore, gene expression (6). In most species, histone H2B is primarily acetylated at Lys5, 12, 15, and 20 (4,7). Histone H3 is primarily acetylated at Lys9, 14, 18, 23, 27, and 56. Acetylation of H3 at Lys9 appears to have a dominant role in histone deposition and chromatin assembly in some organisms (2,3). Phosphorylation at Ser10, Ser28, and Thr11 of histone H3 is tightly correlated with chromosome condensation during both mitosis and meiosis (8-10). Phosphorylation at Thr3 of histone H3 is highly conserved among many species and is catalyzed by the kinase haspin. Immunostaining with phospho-specific antibodies in mammalian cells reveals mitotic phosphorylation at Thr3 of H3 in prophase and its dephosphorylation during anaphase (11).染色质结构调整在真核生物的转录调控中扮演着一个重要角色。核小体是染色质的基本结构单位，由DNA缠绕八个核心组蛋白 (H2A、H2B、H3、H4各2个分子)组成(1)。核心组蛋白的氨基端尾部常发生多种翻译后修饰，包括乙酰化、磷酸化、甲基化和泛素化 (2-5)。这些修饰都是针对不同刺激而产生的反应，且会直接影响染色质对转录因子的可接近性，进而影响基因表达（6）。在多数物种中，组蛋白H2B 乙酰化主要发生在Lys5， 12， 15和20位点上 (4,7)。而组蛋白H3的乙酰化则主要发生在Lys9， 14， 18， 23， 27和56位点上。在一些生物体内，H3 Lys9位点的乙酰化修饰看似对组蛋白沉积和染色质组装有决定性的作用 (2,3)。组蛋白H3 Ser10、Ser28和Thr11位点上的磷酸化修饰与有丝分裂和减数分裂期间的染色体浓缩紧密相关(8-10)。组蛋白H3 Thr3位点上的磷酸化在许多物种中是高度保守的，由激酶haspin催化完成。在哺乳动物细胞内用磷酸化特异性抗体进行免疫染色，发现有丝分裂中H3 Thr3 位点的磷酸化发生在前期，而去磷酸化则发生在后期（11）。 |
| 存放说明 | 4C |
| 参考文献 | Workman, J.L. and Kingston, R.E. (1998) Annu Rev Biochem 67, 545-79. Hansen, J.C. et al. (1998) Biochemistry 37, 17637-41. Strahl, B.D. and Allis, C.D. (2000) Nature 403, 41-5. Cheung, P. et al. (2000) Cell 103, 263-71. Bernstein, B.E. and Schreiber, S.L. (2002) Chem Biol 9, 1167-73. Jaskelioff, M. and Peterson, C.L. (2003) Nat Cell Biol 5, 395-9. Thorne, A.W. et al. (1990) Eur J Biochem 193, 701-13. Hendzel, M.J. et al. (1997) Chromosoma 106, 348-60. Goto, H. et al. (1999) J Biol Chem 274, 25543-9. Preuss, U. et al. (2003) Nucleic Acids Res 31, 878-85. Dai, J. et al. (2005) Genes Dev 19, 472-88. |
Figure 1. Treatment of NIH/3T3 cells with trichostatin A (TSA) increases the acetylation of Histone H4 at Lys12 detected by PathScan® Acetyl-Histone H4 (Lys12) Sandwich ELISA Kit #7228. TSA treatment does not affect the level of histone H4 that is detected by Western analysis. NIH/3T3 cells (70-80% confluent) were treated for 16-18 hours with 0.4 μM TSA at 37ºC. Absorbance readings at 450 nm are shown in the top figure while the corresponding Western blots using Histone H4 Antibody #2592 (left panel) or Acetyl-Histone H4 (Lys12) Antibody #2591 (right panel) are shown in the bottom figure.图1：曲古抑菌素A （TSA）处理NIH/3T3后用PathScan® Acetyl-Histone H4 (Lys12) Sandwich ELISA Kit #7228.进行检测，发现组蛋白 H4 Lys8的乙酰化水平明显提高。但Western 检测后发现，TSA处理不会影响组蛋白H4的总水平。将培养至70-80%汇合的NIH/3T3细胞，用0.4 μM TSA37ºC处理16-18个小时。450mn的吸光值见上图， Western blot检测结果见下图。Western使用的抗体是Histone H4 Antibody#2592 (左下图) 或Acetyl-Histone H4 (Lys12) Antibody #2591 (右下图)。 | |
Figure 2: The relationship between protein concentration of lysates from untreated and TSA treated HeLa cells and kit assay optical density readings. HeLa cells were treated with TSA (4μM overnight). An acid extraction was performed for cell lysis in the presence of 5mM sodium butyrate.图2：未处理和经TSF处理后HeLa细胞蛋白浓度与450nm吸光值间关系如图所示。HeLa细胞经TSA处理（4uM 过夜）。在细胞裂解物中加入丁酸钠后进行酸抽提。 |
