Phospho-Chk1/2 Antibody Sampler Kit

• 产品编号：CST-9931S      品牌：Cell Signaling Technology       原厂货号：9931S
• 产品分类：抗体 > 一抗 > 复合抗体试剂盒
• 应用分类：

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描述：

每个磷酸化特异性Chk1抗体都能够检测内源性特定丝氨酸（296位、317位、345位）磷酸化的Chk1蛋白，每个磷酸化特异性Chk2抗体都能够检测内源性特定丝氨酸（19位、33/35位、516位）和苏氨酸（68位）磷酸化的Chk2蛋白。Chk1 Antibody #2345和Chk2 Antibody #2662能够分别检测内源性Chk1和Chk2总蛋白。单克隆抗体是由合成的人源的针对Chk1蛋白丝氨酸（345位）的磷酸化肽段免疫动物生产的。多克隆抗体是由合成的人源的针对Chk1蛋白丝氨酸（296位、317位）的磷酸化肽段和针对Chk2蛋白丝氨酸（19位、33/35位、516位）和苏氨酸（68位）的磷酸化肽段免疫动物生产的。Chk1 Antibody #2345是由合成的人源的针对Chk1蛋白丝氨酸（296位）的肽段免疫动物生产的。Chk2 Antibody #2662是由合成的人源的针对Chk2蛋白氨基末端的肽段免疫动物生产的。多克隆抗体是采用蛋白A和多肽亲和层析技术纯化生产的。Phospho-Chk1/2 Antibody Sampler Kit能够经济地评价Chk1和Chk2蛋白在多位点的磷酸化状态。该试剂盒包含足够四次western blot所需的一抗和二抗。Chk1激酶充当ATM/ATR激酶的下游，在DNA损伤检验点的控制、胚胎发育和肿瘤抑制中扮演着重要角色(1)。Chk1的激活涉及Ser317和Ser345的磷酸化，其发生响应于DNA复制中断和某些形式的基因毒性应激(2)。Ser345的磷酸化有助于检验点激活后Chk1定位到细胞核(3)，而Ser317的磷酸化随伴PTEN的位点特异性磷酸化并允许DNA复制停滞后再次进入细胞周期(4)。Chk1的细胞周期检验点机制的发挥部分通过调节磷酸酶的cdc25家族。Chk1磷酸化cdc25A靶向其蛋白水解并通过14-3-3结合抑制其活性(5)。活化的Chk1可以通过磷酸化Ser216灭活cdc25C，阻断cdc2的激活有丝分裂期的转换(6)。中心体Chk1已被证明能够磷酸化cdc25B和抑制其CDK1细胞周期素B1的激活，从而废除有丝分裂纺锤体的形成和染色质聚集(7)。此外，通过调节极光激酶B和BubR1，Chk1在纺锤体检验点的功能中起到了重要作用。由于Chk1的抑制作用可导致许多肿瘤细胞株死亡，它已成为用于癌症治疗的药物靶标(9)。Chk2是芽殖酵母Rad53和裂殖酵母Cds1检验点激酶在哺乳动物中的同源蛋白(5-7)。Chk2的氨基末端结构域包含一个7丝氨酸或苏氨酸残基系列(Ser19, Thr26, Ser28, Ser33, Ser35, Ser50和Thr68)，每个紧接着谷氨酰胺(SQ或 TQ基序)。这些位点被认为是ATM/ATR激酶磷酸化的从优位置(8)。电离辐射(IR)，紫外光照射或羟基脲处理引起DNA损伤后，该区域的Thr68和其它位点被ATM/ ATR磷酸化(9-11)。因此，SQ/TQ簇结构域似乎有调节功能。Thr68磷酸化是随后活化步骤的一个先决条件，这有助于激酶结构域活化回路Chk2 的Thr383和Thr387残基发生自身磷酸化(12)。

1. Liu, Q. et al. (2000) Genes Dev 14, 1448-59.
2. Zhao, H. and Piwnica-Worms, H. (2001) Mol Cell Biol 21, 4129-39.
3. Jiang, K. et al. (2003) J Biol Chem 278, 25207-17.
4. Martin, S.A. and Ouchi, T. (2008) Mol Cancer Ther 7, 2509-16.
5. Chen, M.S. et al. (2003) Mol Cell Biol 23, 7488-97.
6. Zeng, Y. et al. (1998) Nature 395, 507-10.
7. Löffler, H. et al. (2006) Cell Cycle 5, 2543-7.
8. Zachos, G. et al. (2007) Dev Cell 12, 247-60.
9. Garber, K. (2005) J Natl Cancer Inst 97, 1026-8.
10. Allen, J. B. et al. (1994) Genes Dev. 8, 2401-2415.
11. Weinert, T. A. et al. (1994) Genes Dev. 8, 652-665.
12. Murakami, H. and Okayama, H. (1995) Nature 374, 817-819.
13. Kastan, M.B. and Lim, D.S. (2000) Nat. Rev. Mol. Cell Biol. 1, 179-186.
14. Matsuoka, S. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 10389-10394.
15. Melchionna, R. et al. (2000) Nat. Cell Biol. 2, 762-765.
16. Ahn, J. Y. et al. (2000) Cancer Res. 60, 5934-5936.

原厂资料：

Specificity / Sensitivity

Each phospho-specific Chk1 antibody detects endogenous levels of Chk1 when phosphorylated at the indicated site (Ser296, Ser317 or Ser345), and each phospho-specific Chk2 antibody detects endogenous levels Chk2 only when phosphorylated at the indicated site (Ser19, Ser33/35, Thr68, and Ser516). Chk1 Antibody #2345 detects endogenous levels of total Chk1. Chk2 Antibody #2662 detects endogenous levels of total Chk2.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser345 of human Chk1. Polyclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Ser296 or Ser317 of human Chk1 and Ser19, Ser33/35, Thr68, Ser516 of human Chk2. Chk1 Antibody #2345 is produced by immunizing rabbits with a synthetic peptide corresponding to residues around Ser296 of human Chk1. Chk 2 Antibody #2662 is produced by immunizing rabbits with a synthetic peptide corresponding to amino-terminal residues of human Chk2. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.

Description

The Phospho-Chk1/2 Antibody Sampler Kit offers an economical means to evaluate the phosphorylation status of Chk1 and Chk2 on multiple residues. The kit contains enough primary and secondary antibodies to perform four Western blot experiments with each primary antibody.

Background

Chk1 kinase acts downstream of ATM/ATR kinase and plays an important role in DNA damage checkpoint control, embryonic development, and tumor suppression (1). Activation of Chk1 involves phosphorylation at Ser317 and Ser345 and occurs in response to blocked DNA replication and certain forms of genotoxic stress (2). While phosphorylation at Ser345 serves to localize Chk1 to the nucleus following checkpoint activation (3), phosphorylation at Ser317 along with site-specific phosphorylation of PTEN allows for reentry into the cell cycle following stalled DNA replication (4). Chk1 exerts its checkpoint mechanism on the cell cycle, in part, by regulating the cdc25 family of phosphatases. Chk1 phosphorylation of cdc25A targets it for proteolysis and inhibits its activity through 14-3-3 binding (5). Activated Chk1 can inactivate cdc25C via phosphorylation at Ser216, blocking the activation of cdc2 and transition into mitosis (6). Centrosomal Chk1 has been shown to phosphorylate cdc25B and inhibit its activation of CDK1-cyclin B1, thereby abrogating mitotic spindle formation and chromatin condensation (7). Furthermore, Chk1 plays a role in spindle checkpoint function through regulation of Aurora B and BubR1 (8). Chk1 has emerged as a drug target for cancer therapy as its inhibition leads to cell death in many cancer cell lines (9). Chk2 is the mammalian homologue of the budding yeast Rad53 and fission yeast Cds1 checkpoint kinases (5-7). The amino-terminal domain of Chk2 contains a series of seven serine or threonine residues (Ser19, Thr26, Ser28, Ser33, Ser35, Ser50 and Thr68) followed by glutamine (SQ or TQ motif). These are known to be preferred sites for phosphorylation by ATM/ATR kinases (8). Indeed, after DNA damage by ionizing radiation (IR), UV irradiation and DNA replication blocked by hydroxyurea, Thr68 and other sites in this region become phosphorylated by ATM/ATR (9-11). The SQ/TQ cluster domain, therefore, seems to have a regulatory function. Phosphorylation at Thr68 is a prerequisite for the subsequent activation step, which is attributable to autophosphorylation of Chk2 on residues Thr383 and Thr387 in the activation loop of the kinase domain (12). 

1. Liu, Q. et al. (2000) Genes Dev 14, 1448-59.
2. Zhao, H. and Piwnica-Worms, H. (2001) Mol Cell Biol 21, 4129-39.
3. Jiang, K. et al. (2003) J Biol Chem 278, 25207-17.
4. Martin, S.A. and Ouchi, T. (2008) Mol Cancer Ther 7, 2509-16.
5. Chen, M.S. et al. (2003) Mol Cell Biol 23, 7488-97.
6. Zeng, Y. et al. (1998) Nature 395, 507-10.
7. Löffler, H. et al. (2006) Cell Cycle 5, 2543-7.
8. Zachos, G. et al. (2007) Dev Cell 12, 247-60.
9. Garber, K. (2005) J Natl Cancer Inst 97, 1026-8.
10. Allen, J. B. et al. (1994) Genes Dev. 8, 2401-2415.
11. Weinert, T. A. et al. (1994) Genes Dev. 8, 652-665.
12. Murakami, H. and Okayama, H. (1995) Nature 374, 817-819.
13. Kastan, M.B. and Lim, D.S. (2000) Nat. Rev. Mol. Cell Biol. 1, 179-186.
14. Matsuoka, S. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 10389-10394.
15. Melchionna, R. et al. (2000) Nat. Cell Biol. 2, 762-765.
16. Ahn, J. Y. et al. (2000) Cancer Res. 60, 5934-5936.

注意事项：

Storage: Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM
NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not
aliquot the antibodies.