| 货号 | 7253C |
| 描述 | The PathScan® Total Histone H3 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of histone H3. A Histone H3 Rabbit Antibody* has been coated onto the microwells. After incubation with cell lysates, histone H3 (modified and unmodified) is captured by the coated antibody. Following extensive washing, biotinylated Histone H3 Rabbit Antibody* is added to detect the captured histone H3 protein. HRP-linked streptavidin 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 total histone H3. * Antibodies in kit are custom formulations specific to kit. |
| 反应种属 | Human/Mouse/Monkey |
| 应用 | ELISA |
| 目标/特异性 | CSTs PathScan® Total Histone H3 Sandwich ELISA Kit #7253 detects endogenous levels of histone H3. As shown in Figure 1, using the Total Histone H3 Sandwich ELISA Kit #7253, a high level of histone H3 is detected in NIH/3T3 cells when treated with TSA. The level of total histone H3 (modified and unmodified) 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). Note: For this assay, it is recommended that lysates be thoroughly sonicated to ensure complete extraction of histone H3 and an accurate absorbance reading. 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). |
| 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 H3 at Lys9, detected by PathScan® Acetyl-Histone H3 (Lys9) Sandwich ELISA Kit #7121, and the di-methylation of histone H3 at Lys4, detected by PathScan® Di-Methyl-Histone H3 (Lys4) Sandwich ELISA Kit #7124. However, TSA treatment does not affect the level of total histone H3 that is detected by Pathscan® Total Histone H3 Sandwich ELISA Kit #7253. 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 H3 Antibody #9715 (left panel), Acetyl-Histone H3 (Lys9) Antibody #9671 (middle panel) or Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb #9725 (right panel) are shown in the bottom figure. 图1：使用trichostatin A (TSA)刺激NIH/3T3细胞系，使得histone H3蛋白的Lys 9位点乙酰化的水平升高，随后用PathScan® Acetyl-Histone H3 (Lys9) Sandwich ELISA Kit #7121检测，再通过PathScan® Di-Methyl-Histone H3 (Lys4) Sandwich ELISA Kit #7124检测Histone H3蛋白的Lys4位点的双甲基化。然而，TSA处理不影响histone H3的总蛋白水平，通过Pathscan® Total Histone H3 Sandwich ELISA Kit #7253检测。NIH/3T3细胞(70-80% confluent)在37ºC条件下使用0.4 μM TSA处理16-18小时。在450 nm吸光度值显示在最上图中，然而通过使用Histone H3 Antibody #9715 (左图), Acetyl-Histone H3 (Lys9) Antibody #9671 (中图)或Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb #9725 (右图)，相应的免疫印迹（western blot）显示在下图中。 | |
Figure 2. The relationship between the protein concentration of the lysate from untreated and TSA-treated NIH/3T3 cells and the absorbance at 450 nm is shown. 图2：未处理和TSA处理了的NIH/3T3细胞裂解的蛋白浓度与450 nm的吸光度读数的关系。 | |
Figure 3. Kit specificity as demonstrated by Western analysis of the ELISA microwell captured protein. Lysates were prepared from NIH/3T3 cells and incubated in microwells coated with the Histone H3 capture antibody. Wells were washed, and the captured protein was solubilized in SDS gel loading buffer. Western analysis of NIH/3T3 cell starting lysate (lanes 1 & 2) and the captured protein (lanes 3 & 4) was performed using Histone H3 Antibody #9715. The major band detected in the captured material corresponds to histone H3 (lanes 3 & 4). 图3. 该试剂盒的特异性通过ELISA微孔捕捉蛋白的免疫印迹实验确定。NIH/3T3细胞被裂解，并且在微孔内与已固定的Histone H3 capture antibody孵育。微孔被清洗，并且被捕获的蛋白质被溶解在SDS胶的上样缓冲液中。使用Histone H3 Antibody #9715，免疫印迹分析NIH/3T3细胞开始裂解液(第1和2道)和捕获的蛋白(第3和4道)。在捕获的蛋白液中主要的条带被检测，其与Histone H3相对应(第3和4道)。 |
