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PathScan® Acetylated p53 Sandwich ELISA Kit #7236

cell based assay   cell-based assay   p53 elisa   sandwich elisa   screening  

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No. Size Price
7236C 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
p53 Mouse mAb coated microwells 96 tests
Sealing Tape 2 sheets

Specificity / Sensitivity

CST's PathScan® Acetylated p53 Sandwich ELISA Kit detects endogenous levels of Acetylated p53. Using this Sandwich ELISA Kit #7236, acetylated lysines on p53 are detected when treated with TSA in COS cells. However, the levels of p53 remain unchanged, as shown by Western analysis (Figure 1). NIH/3T3 and 293 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's PathScan® Acetylated p53 Sandwich ELISA Kit能够检测内源性乙酰化p53蛋白。使用Sandwich ELISA Kit #7236,能够在TSA处理的COS细胞中检测到赖氨酸乙酰化p53蛋白。然而,western blot方法检测的p53蛋白没有改变(如图1)。该试剂盒能够检测通过内部测试的特定种属的蛋白,但是也有可能能够检测其它种属的同源蛋白。

Description

The PathScan® Acetylated p53 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of acetylated lysines on p53. A p53 Rabbit Antibody* has been coated onto the microwells. After incubation with cell lysates, the p53 is captured by the coated antibody. Following extensive washing, Acetylated-Lysine Mouse Antibody* is added to detect the acetylated lysines on the p53 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. HRP substrate, TMB is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of acetylated p53. 
 * Antibodies in kit are custom formulations specific to kit.CST's PathScan® Acetylated p53 Sandwich ELISA Kit是一种固相酶联免疫吸附试剂盒,能够检测内源性赖氨酸乙酰化的p53蛋白的水平。微孔板上已包被有一种Rabbit Antibody*。细胞裂解液孵育之后,p53蛋白被包被抗体捕获。洗涤之后,加入Acetylated-Lysine Mouse Antibody*检测已捕获的赖氨酸乙酰化p53蛋白。使用HRP-linked anti-mouse antibody #7076*识别联胞检测抗体。加入HRP底物(TMB)进行显色。显色过程中吸光度的强度和乙酰化p53蛋白的量成正比。该试剂盒中的抗体具有特异性的固定组成。

ELISA - Western correlation

ELISA - Western correlation

Figure 1: Treatment of COS cells with TSA causes accumulation of acetylation on p53, detected by Sandwich ELISA Kit #7236, but does not affect the level of p53 protein, detected by Western analysis. The absorbance at 450 nm is shown in the top figure, while the corresponding Western blot using the Acetylated Lysine mouse mAb (Ac-K-103) #9681 (left panel) or p53 Antibody #2524 (right panel), is shown in the bottom figure.图1.TSA处理HT-29细胞引起p53蛋白乙酰化累积,使用Sandwich ELISA Kit #7236检测,但并不影响p53蛋白的水平,总蛋白水平由western blot检测。上图示, 450nm处的吸光值,相应的western blot检测见下图,使用的抗体为Acetylated Lysine mouse mAb (Ac-K-103) #9681 (左侧) 或p53 Antibody #2524 (右侧)。

Sensitivity

Sensitivity

Figure 2: The relationship between protein concentration of lysates from untreated and TSA treated COS cells and the absorbance at 450 nm is shown. COS cells (80% confluence) were treated with TSA (0.4 μM overnight).图2.TSA处理和非处理COS细胞裂解液蛋白浓度和450nm处吸光值之间的线性关系。TSA (0.4 μM)过夜处理COS细胞(80%融合度)。

Background

The p53 tumor suppressor protein plays a major role in cellular response to DNA damage and other genomic aberrations. Activation of p53 can lead to either cell cycle arrest and DNA repair or apoptosis (1). p53 is phosphorylated at multiple sites in vivo and by several different protein kinases in vitro (2,3). DNA damage induces phosphorylation of p53 at Ser15 and Ser20 and leads to a reduced interaction between p53 and its negative regulator, the oncoprotein MDM2 (4). MDM2 inhibits p53 accumulation by targeting it for ubiquitination and proteasomal degradation (5,6). p53 can be phosphorylated by ATM, ATR, and DNA-PK at Ser15 and Ser37. Phosphorylation impairs the ability of MDM2 to bind p53, promoting both the accumulation and activation of p53 in response to DNA damage (4,7). Chk2 and Chk1 can phosphorylate p53 at Ser20, enhancing its tetramerization, stability, and activity (8,9). p53 is phosphorylated at Ser392 in vivo (10,11) and by CAK in vitro (11). Phosphorylation of p53 at Ser392 is increased in human tumors (12) and has been reported to influence the growth suppressor function, DNA binding, and transcriptional activation of p53 (10,13,14). p53 is phosphorylated at Ser6 and Ser9 by CK1δ and CK1ε both in vitro and in vivo (13,15). Phosphorylation of p53 at Ser46 regulates the ability of p53 to induce apoptosis (16). Acetylation of p53 is mediated by p300 and CBP acetyltransferases. Inhibition of deacetylation suppressing MDM2 from recruiting HDAC1 complex by p19 (ARF) stabilizes p53. Acetylation appears to play a positive role in the accumulation of p53 protein in stress response (17). Following DNA damage, human p53 becomes acetylated at Lys382 (Lys379 in mouse) in vivo to enhance p53-DNA binding (18). Deacetylation of p53 occurs through interaction with the SIRT1 protein, a deacetylase that may be involved in cellular aging and the DNA damage response (19). p53肿瘤抑制蛋白在细胞响应DNA损伤和其它基因组异常的过程中发挥重要作用。p53的激活能够引起细胞周期捕获和DNA修复或细胞凋亡(1)。离体或者在体情况下,p53可以在多个位点被几个蛋白激酶进行磷酸化(2,3)。DNA损伤能够诱导p53的第15位和20位丝氨酸磷酸化,导致p53和其负调节子-癌蛋白MDM2的相互作用减弱(4)。MDM2通过靶向p53促进其泛素化和蛋白酶体降解抑制其累积。P53的第15位和37位丝氨酸可以被ATM,ATR,和DNA-PK磷酸化(5,6)。P53的磷酸化削弱了MDM2的结合,从而促进了它的激活和累积以响应DNA损伤(4,7)。Chk2和Chk1能够磷酸化p53的第20位丝氨酸,增强其四聚化、稳定性和活性(8,9)。p53的第392位丝氨酸可以发生在体磷酸化(10,11)和CAK介导的离体磷酸化(11)。人类肿瘤中p53的第392位丝氨酸磷酸化增加(12)且有报道认为该过程能够影响生长抑制剂的功能、DNA结合和p53的转录激活(10,13,14)。P53的第6位和9位丝氨酸离体或者在体均可以被CK1δ和CK1ε磷酸化(13,15)。P53的第46位丝氨酸磷酸化能够调节p53诱导细胞凋亡的能力(16)。p300和CBP乙酰转移酶能够介导p53的乙酰化。去乙酰化抑制可以阻止MDM2招募p19 (ARF)介导的HDAC1复合体从而稳定p53。乙酰化似乎对于应激反应中p53蛋白的累积起着正向的作用(17)。DNA损伤后,人类p53的第382位赖氨酸(在小鼠为379位赖氨酸)发生在体乙酰化,促进p53-DNA的结合(18)。P53通过与SIRT1蛋白(一种参与细胞衰老和DNA损伤应激的去乙酰酶)相互作用发生去乙酰化(19)。

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

PathScan is a trademark of Cell Signaling Technology, Inc.

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