PathScan? Acetyl-Histone H2B Sandwich ELISA Kit

• 产品编号：CST-7178C      品牌：Cell Signaling Technology       原厂货号：7178C
• 产品分类：ELISA及其它免疫分析 > ELISA > ELISA检测试剂盒
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描述：

CST's PathScan® Acetyl-Histone H2B Sandwich ELISA Kit能够检测乙酰化的内源性histone H2B蛋白水平。使用Sandwich ELISA Kit #7178，当COS细胞中使用TSA处理后，Histone H2B的乙酰化残基能被检测。然而，使用Histone H2B Antibody #2722，histone H2B总蛋白水平通过免疫印迹（western blot）分析仍然没有改变(图1)。当Jurkat 细胞使用TSA处理后同样的结果会出现(数据未给出)。通过内部验证，该试剂盒可以检测已列出的物种，但是可能也会检测其它物种的同源性蛋白。PathScan® Acetyl-Histone H2B (Lys20) Sandwich ELISA Kit是一个固相夹心酶联免疫吸附法，其能检测乙酰化的内源性Histone H2B蛋白。一个Histone H2B Antibody已经被包被在微孔板上。在与细胞裂解液一起孵化后，histone H2B蛋白被包被的抗体吸附。在大范围清洗之后，加入Acetylated-Lysine Mouse mAb鼠单抗检测已被吸附的乙酰化的histone H2B蛋白。然后，anti-mouse IgG, HRP偶联抗体作为二抗来识别已结合的检测抗体。HRP底物（TMB）加入后显色。该显色的吸光度值的大小与乙酰化的histone H2B蛋白量成正比例。试剂盒里的抗体是针对该试剂盒特制的。染色质结构的修饰在调节真核细胞的转录中扮演着重要的角色。由DNA围绕和八聚体组蛋白（H2A，H2B，H3和H4各两个）共同组成的核小体是染色质的主要组成（1）。核小体的组蛋白氨基酸尾端进行不同的转录后修饰，包括乙酰化，磷酸化，甲基化和泛素化（2-5）。这些修饰通过不同的刺激产生并且对转录因子能否接近染色质有着直接的影响，所以也影响着基因的表达（6）。在大多数的物种中组蛋白H2B主要在Lys5,，12,，15和20位点上发生乙酰化（4,7）。组蛋白H3主要是在Lys9，14，18，23，27和56位点上发生乙酰化。在某些物种里H3上的Lys9位点发生乙酰化并应该在组蛋白沉积和染色质组装中扮演着重要的角色（2,3）。组蛋白H3上Ser10，Ser28和Thr11位点的磷酸化在有丝分裂和无丝分裂中都与染色质的缩合紧密相连（8-10）。H3的Thr3位点的磷酸化在许多物种中都是高度保守的，是由kinase haspin所催化的。在哺乳动物中用磷酸化特异性的抗体做免疫组化显示H3的Thr3位点在有丝分裂的前期发生磷酸化，后期发生去磷酸化（11）。

1. Workman, J.L. and Kingston, R.E. (1998) Annu Rev Biochem 67, 545-79.
2. Hansen, J.C. et al. (1998) Biochemistry 37, 17637-41.
3. Strahl, B.D. and Allis, C.D. (2000) Nature 403, 41-5.
4. Cheung, P. et al. (2000) Cell 103, 263-71.
5. Bernstein, B.E. and Schreiber, S.L. (2002) Chem Biol 9, 1167-73.
6. Jaskelioff, M. and Peterson, C.L. (2003) Nat Cell Biol 5, 395-9.
7. Thorne, A.W. et al. (1990) Eur J Biochem 193, 701-13.
8. Hendzel, M.J. et al. (1997) Chromosoma 106, 348-60.
9. Goto, H. et al. (1999) J Biol Chem 274, 25543-9.
10. Preuss, U. et al. (2003) Nucleic Acids Res 31, 878-85.
11. Dai, J. et al. (2005) Genes Dev 19, 472-88.

原厂资料：

Specificity / Sensitivity

CST's PathScan® Acetyl-Histone H2B Sandwich ELISA Kit detects endogenous levels of acetylated Histone H2B. Using this Sandwich ELISA Kit #7178, acetylated lysines on Histone H2B are detected when treated with TSA in COS cells. However, the levels of total Histone H2B protein remain unchanged, as shown by Western blot analysis, using the Histone H2B Antibody #2722 (figure 1). Jurkat cells treated with TSA show similar results (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.

Description

The PathScan® Acetyl-Histone H2B Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of acetylated lysines on Histone H2B. A Histone H2B Antibody has been coated onto the microwells. After incubation with cell lysates, Histone H2B is captured by the coated antibody. Following extensive washing, an Acetylated-Lysine Mouse mAb is added to detect the acetylated lysines on the Histone H2B protein. Anti-mouse IgG, HRP linked Antibody #7076 is then used to recognize the bound detection antibody. HRP substrate, TMB is added to develop color. The magnitude of optical density for this developed color is proportional to the quantity of acetylated Histone H2B. 
 * Antibodies in kit are custom formulations specific to kit.

Background

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).

1. Workman, J.L. and Kingston, R.E. (1998) Annu Rev Biochem 67, 545-79.
2. Hansen, J.C. et al. (1998) Biochemistry 37, 17637-41.
3. Strahl, B.D. and Allis, C.D. (2000) Nature 403, 41-5.
4. Cheung, P. et al. (2000) Cell 103, 263-71.
5. Bernstein, B.E. and Schreiber, S.L. (2002) Chem Biol 9, 1167-73.
6. Jaskelioff, M. and Peterson, C.L. (2003) Nat Cell Biol 5, 395-9.
7. Thorne, A.W. et al. (1990) Eur J Biochem 193, 701-13.
8. Hendzel, M.J. et al. (1997) Chromosoma 106, 348-60.
9. Goto, H. et al. (1999) J Biol Chem 274, 25543-9.
10. Preuss, U. et al. (2003) Nucleic Acids Res 31, 878-85.
11. Dai, J. et al. (2005) Genes Dev 19, 472-88.

注意事项：

* 12 8-well modules -Each module is designed to break apart for 8 tests.
**Kit should be stored at 4°C with the exception of 10X Cell Lysis Buffer, which is stored at –20°C (packaged separately).