Cell Signaling Technology

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PathScan® Acetyl-Histone H2A Sandwich ELISA Kit #7233

Cell based assay   cell-based assay   Histone elisa   histone h2a elisa   sandwich elisa   screening  

REACTIVITY
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No. Size Price
7233C 1 Kit ( 96 assays ) ¥6,345.00 现货查询 购买询价
Product Includes Volume Solution Color
ELISA Sample Diluent 25 ml Blue
Anti-rabbit IgG, HRP-linked Antibody (ELISA Formulated) 1 ea Red (Lyophilized)
Detection Antibody Diluent 11 ml Green
HRP Diluent 11 ml Red
Acetylated-Lysine Rabbit Detection mAb 1 ea Green (Lyophilized)
STOP Solution #7002 11 ml Colorless
TMB Substrate #7004 11 ml Colorless
ELISA Wash Buffer (20X) 25 ml Colorless
Cell Lysis Buffer (10X) #9803 15 ml Yellowish
Histone H2A Ab CoatedMicrowells 96 tests
Sealing Tape 2 sheets

Specificity / Sensitivity

CST's PathScan® Acetyl-Histone H2A Sandwich ELISA Kit detects endogenous levels of Acetylated Histone H2A. Using this Sandwich ELISA Kit #7233, acetylated lysines on Histone H2A are detected when treated with TSA in Jurkat cells. However, the levels of Histone H2A remain unchanged, as shown by Western analysis using the Histone H2A Antibody #2578 (figure 1). COS and NIH 3T3 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.

CST的PathScan®Acetyl-Histone H2A Sandwich ELISA Kit 能够检测内源性乙酰化组蛋白H2A的水平。使用该Sandwich ELISA Kit #7233可以检测到经TSA处理过的Jurkat细胞内组蛋白H2A 上乙酰化的赖氨酸。而当使用Histone H2A Antibody #2578 进行Western 检测,显示组蛋白H2A总水平保持不变(图1)。当检测经TSA处理的COS和NIH/3T3细胞时也得到了类似的结果(数据未列出)。经内部测试确认,本试剂盒可以检测到所列物种中的蛋白。但对其他物种中的同源蛋白可能也可以检测。

Description

CST's PathScan® Acetyl-Histone H2A Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of acetylated lysines on Histone H2A. A Histone H2A Antibody has been coated onto the microwells. After incubation with cell lysates, Histone H2A is captured by the coated antibody. Following extensive washing, Acetylated-Lysine Rabbit mAb (Ac-K2-100) is added to detect the acetylated lysines on the Histone H2A 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 optical density for this developed color is proportional to the quantity of acetylated Histone H2A. 
 Antibodies in kit are custom formulations specific to kit.

PathScan® Acetyl-Histone H2A Sandwich ELISA Kit是通过固相夹心酶联免疫吸附法(ELISA)来检测内源性的组蛋白H2A 上乙酰化赖氨酸水平的。先将组蛋白H2A小鼠抗体包被在微孔板上。与细胞裂解物孵育后,组蛋白H2A便可被包被的抗体捕获。清洗彻底后,加入Acetylated-Lysine Rabbit mAb (Ac-K2-100) 检测组蛋白H2A 蛋白上的乙酰化赖氨酸。再加入HRP标记抗兔 IgG作为二抗来识别之前绑定的检测抗体。最后加入HRP底物—— TMB,进行显色。产生的吸光值与乙酰化组蛋白H2A的数量成正比。试剂盒中抗体是专为本试剂盒特制的。

Sandwich ELISA

Sandwich ELISA

Figure 1: Treatment of Jurkat cells with TSA causes accumulation of acetylation on Histone H2A, detected by Sandwich ELISA Kit, #7233, but does not affect the level of total Histone H2A protein, detected by Western analysis. OD 450nm readings are shown in the top figure, while the corresponding Western blot using the Acetylated Lysine mouse mAb (Ac-K-103) #9681 (left panel) or Histone H2A Antibody #2578 (right panel), is shown in the bottom figure.图1:使用Sandwich ELISA Kit, #7233检测发现TSA处理Jurkat细胞会引起组蛋白 H2A上乙酰化的积累。但Western 检测后发现,TSA处理不会影响组蛋白H2A的总水平。450mn的吸光值见上图, Western blot检测结果见下图。Western使用的抗体是Acetylated Lysine mouse mAb (Ac-K-103) #9681 (左下图) 或Histone H2A Antibody #2578 (右下图)。

Sensitivity

Sensitivity

Figure 2: The relationship between protein concentration of lysates from untreated and TSA treated COS cells and kit assay optical density readings. COS cells were treated with TSA (4 µM overnight). An acid extraction was performed for cell lysis in the presence of 5 mM sodium butyrate.图2:未处理和经TSF处理后COS细胞蛋白浓度与450nm吸光值间关系如图所示。COS细胞经TSA处理(4uM 过夜)。在细胞裂解物中加入5nM丁酸钠后进行酸抽提。

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).

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)。

  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.

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For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

PathScan is a trademark of Cell Signaling Technology, Inc.

Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.

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