| 货号 | 7037C |
| 描述 | The PathScan® Phospho-Chk2 (Thr68) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Chk2 when phosphorylated at Thr68. A phospho-Chk2 (Thr68) rabbit antibody has been coated onto the microwells. After incubation with cell lysates, phospho-Chk2 protein is captured by the coated antibody. Following extensive washing, a Chk2 mouse detection antibody is added to detect the captured Chk2 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. The magnitude of the absorbance for the developed color is proportional to the quantity of Chk2 phosphorylated at Thr68. Antibodies in kit are custom formulations specific to kit.PathScan® Phospho-Chk2 (Thr68) Sandwich ELISA Kit是一种固相酶联免疫吸附试剂盒，能够检测内源性苏氨酸68位磷酸化的Chk2蛋白。微孔板上已包被有一种phospho-Chk2 (Thr68) rabbit antibody。细胞裂解液孵育之后，phospho-Chk2蛋白被包被抗体捕获。洗涤之后，加入Chk2 mouse detection antibody检测已捕获的Chk2蛋白。使用抗鼠IgG和HRP-连接的抗体#7076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苏氨酸68位磷酸化的Chk2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® Phospho-Chk2 (Thr68) Sandwich ELISA Kit #7037 detects endogenous levels of Chk2 protein when phosphorylated at Thr68. 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 | Chk2 is the mammalian orthologue of the budding yeast Rad53 and fission yeast Cds1 checkpoint kinases (1-3). The amino-terminal domain of Chk2 contains a series of seven serine or threonine residues (Ser19, Thr26, Ser28, Ser33, Ser35, Ser50 and Thr68) each followed by glutamine (SQ or TQ motif). These are known to be preferred sites for phosphorylation by ATM/ATR kinases (4,5). After DNA damage by ionizing radiation (IR), UV irradiation or hydroxyurea treatment, Thr68 and other sites in this region become phosphorylated by ATM/ATR (5-7). 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 (8).Chk2是哺乳动物中和芽殖酵母Rad53和裂殖酵母Cds1检验点激酶同源的蛋白(1-3)。Chk2的氨基末端结构域包含有一个七丝氨酸或者苏氨酸残基(Ser19, Thr26, Ser28, Ser33, Ser35, Ser50 and Thr68)组成的系列，每个系列紧跟着谷氨酸(SQ 或TQ模体)。这些地方是已知的ATM/ATR激酶磷酸化的首选作用位点(4,5)。电离辐射(IR)引起DNA损伤之后，UV照射处理或者羟基脲处理，这个区域的68位苏氨酸和其他位点被ATM/ATR磷酸化(5-7)。因此，SQ/TQ簇结构域似乎具有调节功能。第68位苏氨酸的磷酸化是后续激活步骤的先决条件，激活归因于激酶结构域激活回路中Chk2第383位和387位苏氨酸残基的自体磷酸化(8)。 |
| 存放说明 | 4C |
| 参考文献 | Allen, J.B. et al. (1994) Genes Dev. 8, 2401-2415. Weinert, T.A. et al. (1994) Genes Dev. 8, 652-665. Murakami, H. and Okayama, H. (1995) Nature 374, 817-819. Kastan, M.B. and Lim, D.S. (2000) Nat. Rev. Mol. Cell Biol. 1, 179-186. Matsuoka, S. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 10389-10394. Melchionna, R. et al. (2000) Nat. Cell Biol. 2, 762-765. Ahn, J.Y. et al. (2000) Cancer Res. 60, 5934-5936. Lee, C.H. and Chung, J.H. (2001) J. Biol. Chem. 276, 30537-30541. |
Figure 1. Treatment of HeLa cells with UV stimulates phosphorylation of Chk2 at Thr68, detected by the PathScan® Phospho-Chk2 (Thr68) Sandwich ELISA Kit #7037, but does not affect the levels of total Chk2 detected by PathScan® Total Chk2 Sandwich ELISA Kit #7045. HeLa cells (80-90% confluent) were treated with 100 mJ/cm2 UV with 1 hour recovery at 37º C. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using Chk2 (1C12) Mouse mAb #3440 (left panel) or Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb #2197 (right panel) are shown in the bottom figure. 图1.使用PathScan® Phospho-Chk2 (Thr68) Sandwich ELISA Kit #7037检测紫外刺激诱导HeLa细胞苏氨酸68位磷酸化的Chk2蛋白，但紫外诱导并不影响Chk2总蛋白水平，Chk2总蛋白由PathScan® Total Chk2 Sandwich ELISA Kit #7045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Chk2 (1C12) Mouse mAb #3440 (左下)或Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb #2197 (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® Phospho-Chk2 (Thr68) Sandwich ELISA Kit #7037 is shown.图2,. 未处理和紫外处理的HeLa细胞裂解液蛋白浓度之间的关系，图示为450nm处二者的吸光值，使用PathScan® Phospho-Chk2 (Thr68) Sandwich ELISA Kit #7037进行检测。 |
