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PathScan® Acetyl-Histone H3 (Lys9) Sandwich ELISA Kit #7121

cell based assay   cell-based assay   Histone H3 elisa   sandwich elisa   screening  

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No. Size Price
7121C 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-mouse IgG, HRP-linked Antibody (ELISA Formulated) 1 ea Red (Lyophilized)
Detection Antibody Diluent 11 ml Green
HRP Diluent 11 ml Red
Histone H3 Mouse 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
Sealing Tape 2 sheets
Acetyl-Histone H3 (K9) Rabbit Antibody coated microwells 96 tests

Specificity / Sensitivity

CST's PathScan® Acetyl-Histone H3 (Lys9) Sandwich ELISA Kit #7121 detects endogenous levels of histone H3 when acetylated at Lys9. As shown in Figure 1 using the Acetyl-Histone H3 (Lys9) Sandwich ELISA Kit #7121, a high level of acetylation at Lys9 on histone H3 is detected in COS 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 NIH/3T3 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.CST's

PathScan® Acetyl-Histone H3 (Lys9) Sandwich ELISA Kit #7121能够检测内源性Lys9位点乙酰化的Histone H3的蛋白水平。如图1所示,使用Acetyl-Histone H3 (Lys9) Sandwich ELISA Kit #7121,当COS细胞TSA处理后,Histone H3蛋白Lys9位点的高水平乙酰化被检测。histone H3 (修饰和未修饰)总蛋白水平通过免疫印迹(western blot)分析显示没有变化。使用TSA处理的NIH/3T3和Jurkat细胞显示类似的结果(数据未显示)。因为通过内部实验确定,该试剂盒检测已表明的物种,但是可能也会检测其它物种的同源性蛋白。

Description

The PathScan® Acetyl-Histone H3 (Lys9) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of histone H3 when acetylated at Lys9. An Acetyl-Histone H3 (Lys9) Rabbit Antibody has been coated onto the microwells. After incubation with cell lysates, acetyl-histone H3 (Lys9) is captured by the coated antibody. Following extensive washing, biotinylated Histone H3 Rabbit Antibody is added to detect the 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 histone H3 acetylated at Lys9. 
 Antibodies in kit are custom formulations specific to kit.

该PathScan® Acetyl-Histone H3 (Lys9) Sandwich ELISA Kit是一个固相夹心酶联免疫吸附法,其能检测内源性Lys9位点乙酰化的Histone H3蛋白。一个Acetyl-Histone H3 (Lys9) Rabbit Antibody已经被包被在微孔板上。在与细胞裂解液一起孵化后,acetyl-histone H3 (Lys9)蛋白被包被的抗体吸附。全面清洗之后,一个biotinylated Histone H3 Rabbit Antibody被加入去检测Histone H3蛋白。然后,HRP-linked streptavidin被用于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在Lys9位点乙酰化的Histone H3蛋白量成正比例。试剂盒里的抗体是针对该试剂盒特定制备的。

ELISA - Western correlation

ELISA - Western correlation

Figure 1. Treatment of COS cells with trichostatin A (TSA) increases the acetylation of Histone H3 at Lys9 detected by PathScan® Acetyl-Histone H3 (Lys9) Sandwich ELISA Kit #7121. TSA treatment does not affect the level of histone H3 that is detected by Western analysis. COS 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) or Acetyl-Histone H3 (Lys9) Antibody #9671 (right panel) are shown in the bottom figure.

图1:使用TSA刺激COS细胞系,使用PathScan® Acetyl-Histone H3 (Lys9) Sandwich ELISA Kit #7121进行检测,这增加了histone H3的Lys9乙酰化总蛋白水平。通过免疫印迹检测TSA处理不影响histone H3。COS细胞(70-80% confluent)使用0.4 μM TSA在37ºC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)和Acetyl-Histone H3 (Lys9) Antibody #9671 (右图),相应的免疫印迹(western blot)显示在下图中。

Sensitivity

Sensitivity

Figure 2. The relationship between the protein concentration of the recombinant Histone H3 (Lys9) protein and the absorbance at 450 nm is shown.

图2:该图显示了重组的Histone H3 (Lys9)蛋白浓度与450 nm吸光值读数的关系。

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.

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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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