| 货号 | 7113C |
| 描述 | CST’s PathScan® Phospho-Stat5 (Tyr694) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Stat5a when phosphorylated at Tyr694 and Stat5b at Tyr699. A Phospho-Stat5 Mouse Antibody has been coated onto the microwells. After incubation with cell lysates, phospho-Stat5a (Tyr694) and phospho-Stat5b (Tyr699) proteins are captured by the coated antibody. Following extensive washing, a Stat5 Rabbit Detection Antibody is added to detect the captured phospho-Stat5a and phospho-Stat5b 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 absorbance for this developed color is proportional to the quantity of Stat5a phosphorylated at Tyr694 and Stat5b phosphorylated at Tyr699. Antibodies in kit are custom formulations specific to kit. |
| 反应种属 | Human |
| 应用 | ELISA |
| 目标/特异性 | CST’s PathScan® Phospho-Stat5 (Tyr694) Sandwich ELISA Kit #7113 detects endogenous levels of phospho-Stat5a when phosphorylated at Tyr694 and Stat5b at Tyr699. As shown in Figure 1, a significant induction of Stat5 phosphorylation can be detected in EGF-treated HeLa cells using the Phospho-Stat5 (Tyr694) Sandwich ELISA Kit #7113. The level of total Stat5 detected by western analysis remains unchanged. 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 | Stat5 is activated in response to a wide variety of ligands including IL-2, GM-CSF, growth hormone and prolactin. Phosphorylation at Tyr694 is obligatory for Stat5 activation (1,2). This phosphorylation is mediated by Src upon erythropoietin stimulation (3). Stat5 is constitutively active in some leukemic cell types (4). Phosphorylated Stat5 is found in some endothelial cells treated with IL-3, which suggests its involvement in angiogenesis and cell motility (5). Stat5a and Stat5b are independently regulated and activated in various cell types. For instance, interferon treatment predominantly activates Stat5a in U-937 cells and Stat5b in HeLa cells (6). |
| 存放说明 | 4C |
| 参考文献 | Gouilleux, F. et al. (1994) EMBO J 13, 4361-9. Wakao, H. et al. (1994) EMBO J 13, 2182-91. Okutani, Y. et al. (2001) Oncogene 20, 6643-50. Demoulin, J.B. et al. (1999) J Biol Chem 274, 25855-61. Dentelli, P. et al. (1999) J Immunol 163, 2151-9. Meinke, A. et al. (1996) Mol Cell Biol 16, 6937-44. |
Figure 1. Treatment of HeLa cells with EGF stimulates phosphorylation of Stat5 at Tyr694, detected by the PathScan® Phospho-Stat5 (Tyr694) Sandwich ELISA Kit #7113, but does not affect the level of total Stat5 protein detected by Western analysis. HeLa cells (80-90% confluent) were starved overnight and treated with 100 ng/mL EGF for 40 minutes at 37oC. The absorbance readings at 450 nm are shown in the top figure, while the corresponding Western blots, using Stat5 (3H7) Rabbit mAb #9358 (left panel) or Phospho-Stat5 (Tyr694) (14H2) Mouse mAb #9356 (right panel), are shown in the bottom figure.图1.在HeLa细胞中，用EGF刺激产生Tyr694位点磷酸化的Stat5,并通过PathScan® Phospho-Stat5 (Tyr694) Sandwich ELISA Kit #7113检测, 经western免疫印迹检测表明总Stat5蛋白水平并未受到影响。在37oC下，HeLa细胞(80-90% confluent) 过夜饥饿并用100 ng/mL EGF 处理 40 min。OD450的读数在图片的最上面显示, 同时对应的Western免疫印迹在下面图片显示，所用抗体为Stat5 (3H7) Rabbit mAb #9358 (左侧图) 或 Phospho-Stat5 (Tyr694) (14H2) Mouse mAb #9356 (右侧图)。 | |
Figure 2. The relationship between the protein concentration of the lysate from untreated and EGF-treated HeLa cells and the absorbance at 450 nm is shown.图2.图示为未经处理和经EGF处理的HeLa细胞的裂解液蛋白浓度与450 nm 吸光度的关系。 |
