| 货号 | 7872S |
| 描述 | The PathScan® Total Chk1 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Chk1. A Chk1 Mouse Antibody* has been coated onto the microwells. After incubation with cell lysates, Chk1 (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a Chk1 Rabbit Detection Antibody* is added to the captured phospho and nonphospho Chk1 protein. Anti-rabbit IgG, HRP-linked Antibody #7074* 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 total Chk1. *Antibodies in kit are custom formulations specific to kit.PathScan® Phospho-Aurora A (Thr288) Sandwich ELISA Kit是一种固相酶联免疫吸附试剂盒，能够检测内源性Chk1蛋白。微孔板上已包被有一种Chk1 Mouse Antibody。细胞裂解液孵育之后，Chk1 (磷酸化和非磷酸化)被包被抗体捕获。洗涤之后，加入Chk1 Rabbit Detection Antibody*检测已捕获的磷酸化和非磷酸化Chk1蛋白。使用抗兔IgG和HRP-连接的抗体Chk1 Rabbit Detection Antibody*识别结合的检测抗体。加入HRP底物(TMB)进行显色。显色过程中吸光度的强度和Chk1总蛋白的量成正比。该试剂盒中的抗体具有特异性的固定组成。 |
| 反应种属 | Human |
| 应用 | ELISA |
| 目标/特异性 | CSTs PathScan® Chk1 Sandwich ELISA Kit #7872 detects endogenous levels of total Chk1 protein. As shown in Figure 1, a significant induction of Chk1 phosphorylation at Ser317 can be detected in HeLa cells following treatment with UV using the Phospho-Chk1 (Ser317) Sandwich ELISA Kit #7870. The levels of total Chk1 (phospho and nonphospho) remain unchanged as shown by Western analysis and by PathScan® Total Chk1 Sandwich ELISA Kit #7872 (Figure 1). This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species. |
| 供应商 | CST |
| 背景 | 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).Chk1激酶能够充当ATM/ATR激酶的下游并在DNA损伤检验点控制、胚胎发育和肿瘤抑制中发挥重要作用(1)。Chk1的激活涉及317位和345位丝氨酸的磷酸化，其发生响应DNA复制阻滞和某些类型的基因毒性应激反应(2)。尽管345位丝氨酸的磷酸化有助于伴随检验点激活的Chk1定位到核，317位丝氨酸的磷酸化连同位点特异性的PTEN磷酸化允许DNA复制停滞引起的细胞周期折返(4)。Chk1发挥其细胞周期检验点调节机制部分经由调控磷酸酶家族的cdc25。Chk1磷酸化cdc25A后靶向蛋白降解并通过14-3-3的结合抑制自身的活性(5)。激活的chk1能够通过216位丝氨酸的磷酸化失活cdc25C，阻塞cdc2的激活和有丝分裂转换的进入(6)。中心体的chk1被认为可以磷酸化cdc25B并抑制其激活CDK1-cyclin B1，藉此废除有丝分裂纺锤体的形成和染色质凝聚(7)。此外，Chk1通过调节Aurora B和BubR1在纺锤体检验点功能中发挥重要作用(8)。鉴于Chk1的抑制作用能够引起许多肿瘤细胞系的死亡，它已经成为肿瘤治疗中心的药物靶点(9)。 |
| 存放说明 | 4C |
| 参考文献 | Liu, Q. et al. (2000) Genes Dev 14, 1448-59. Zhao, H. and Piwnica-Worms, H. (2001) Mol Cell Biol 21, 4129-39. Jiang, K. et al. (2003) J Biol Chem 278, 25207-17. Martin, S.A. and Ouchi, T. (2008) Mol Cancer Ther 7, 2509-16. Chen, M.S. et al. (2003) Mol Cell Biol 23, 7488-97. Zeng, Y. et al. (1998) Nature 395, 507-10. Löffler, H. et al. (2006) Cell Cycle 5, 2543-7. Zachos, G. et al. (2007) Dev Cell 12, 247-60. Garber, K. (2005) J Natl Cancer Inst 97, 1026-8. |
Figure 1. Treatment of HeLa cells with UV stimulates phosphorylation of Chk1 at Ser317, detected by the PathScan® Phospho-Chk1 (Ser317) Sandwich ELISA Kit #7870, but does not affect the levels of total Chk1 detected by PathScan® Total Chk1 Sandwich ELISA Kit #7872. HeLa cells (80-90% confluent) were treated with 100 mJ/cm2 UV with 1 hour recovery at 37ºC. The absorbance readings at 450 nm are shown in the top figure, while the corresponding Western blots using Chk1 Antibody #2345 (left panel) or Phospho-Chk1 (Ser317) Antibody #2344 (right panel) are shown in the bottom figure.使用PathScan® Phospho-Chk1 (Ser317) Sandwich ELISA Kit #7870检测紫外刺激诱导HeLa细胞丝氨酸317位磷酸化的Chk1蛋白，但紫外诱导并不影响Chk1总蛋白水平，Chk1总蛋白由PathScan® Total Chk1 Sandwich ELISA Kit #7872检测。100 mJ/cm2 紫外处理HeLa细胞(80-90% 融合度)后，37ºC恢复1小时。上图示450nm处的吸光度，下图为相应的western blot方法验证，使用的是Chk1 Antibody #2345 (左下)或Phospho-Chk1 (Ser317) Antibody #2344 (右下)。 | |
Figure 2. The relationship between the protein concentration of lysates from untreated and UV-treated HeLa cells and the absorbance at 450 nm using the PathScan® Total Chk1 Sandwich ELISA Kit #7872 is shown.未处理和紫外处理的HeLa细胞裂解液蛋白浓度之间的关系，图示450nm处的吸光度，使用PathScan® Total Chk1 Sandwich ELISA Kit #7872 检测。 |
