| 货号 | 7162C |
| 描述 | CSTs PathScan® Total Bad Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total Bad protein. A Bad rabbit mAb* has been coated onto the microwells. After incubation with cell lysates, Bad protein (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a Bad mouse mAb* is added to detect the captured Bad 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 absorbance for this developed color is proportional to the quantity of total Bad protein. * Antibodies in kit are custom formulations specific to kit. |
| 反应种属 | Human/Monkey |
| 应用 | ELISA |
| 目标/特异性 | CSTs PathScan® Total Bad Sandwich ELISA Kit #7162 detects endogenous levels of Bad protein. A significant induction of Bad phosphorylation at Ser112 can be detected in TPA-treated OVCAR8 cells using PathScan® Phospho-Bad (Ser112) Sandwich ELISA Kit #7182. However, the level of total Bad protein (phospho and nonphospho) detected by PathScan®Total Bad Sandwich ELISA Kit #7162 remains unchanged (Figure 1). In Figure 3, Western analysis of protein captured in microwells coated with the Bad antibody shows a major band corresponding to the Bad protein. 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 | Bad is a proapoptotic member of the Bcl-2 family that promotes cell death by displacing Bax from binding to Bcl-2 and Bcl-xL (1,2). Survival factors, such as IL-3, inhibit the apoptotic activity of Bad by activating intracellular signaling pathways that result in the phosphorylation of Bad at Ser112 and Ser136 (2). Phosphorylation at these sites promotes binding of Bad to 14-3-3 proteins to prevent an association between Bad with Bcl-2 and Bcl-xl (2). Akt phosphorylates Bad at Ser136 to promote cell survival (3,4). Bad is phosphorylated at Ser112 both in vivo and in vitro by p90RSK (5,6) and mitochondria-anchored PKA (7). Phosphorylation of Ser155 in the BH3 domain by PKA plays a critical role in blocking the dimerization of Bad and Bcl-xL (8-10). |
| 存放说明 | 4C |
| 参考文献 | Yang, E. et al. (1995) Cell 80, 285-291. Zha, J. et al. (1996) Cell 87, 619-628. Datta, S.R. et al. (1997) Cell 91, 231-241. Peso, L. et al. (1997) Science 278, 687-689. Bonni, A. et al. (1999) Science 286, 1358-1362. Tan, Y. et al. (1999) J. Biol. Chem. 274, 34859-34867. Harada, H. et al. (1999) Mol. Cell 3, 413-422. Tan, Y. et al. (2000) J. Biol. Chem. 275, 25865-25869. Lizcano, J. et al. (2000) Biochem. J. 349, 547-557. Datta, S. et al. (2000) Mol. Cell 6, 41-51. |
Figure 2. The relationship between the protein concentration of untreated and TPA-treated OVCAR8 cell lysates and the absorbance at 450 nm is shown. Cells were serum starved overnight and then treated with 200 nm TPA for 30 min. at 37ºC. 图2. 显示未经处理和经TPA处理的OVCAR8细胞裂解液中蛋白浓度和450 nm处吸光度之间的关系。细胞经过过夜血清饥饿，并在37ºC条件下经200 nm TPA 处理30 min。 | |
Figure 1. Treatment of OVCAR8 cells with TPA stimulates phosphorylation of Bad at Ser112, detected by PathScan® Phospho-Bad (Ser112) Sandwich ELISA Kit #7182, but does not affect the level of total Bad protein detected by PathScan® Total Bad Sandwich ELISA Kit #7162. Lambda phosphatase treatment of control cell lysates (4000 U/mL for 60 minutes at 37ºC) abolishes the basal phosphorylation of Bad as shown by both Sandwich ELISA and Western analysis. The absorbance readings at 450 nm are shown in the top figure, while the corresponding Western blots using Phospho-Bad (Ser112) Antibody #9296 (right panel) or Bad Antibody #9254 (left panel), are shown in the bottom figure. 图1. 用TPA处理OVCAR8细胞，刺激Bad在Ser112位点的磷酸化，然后经过PathScan® Phospho-Bad (Ser112) Sandwich ELISA Kit #7182检测，但是不影响使用PathScan® Total Bad Sandwich ELISA Kit #7162检测 Bad 总蛋白。用Lambda磷酸酶处理对照细胞的裂解液(4000 U/mL ，60分钟，37ºC) 除去了Bad蛋白的基底磷酸化，通过Sandwich ELISA和Western 免疫印迹分析验证)。450 nm的吸光度数值在上图, 相对应的Western blots在下图，所用抗体为Phospho-Bad (Ser112) Antibody #9296 (右图) 或者Bad Antibody #9254 (左图)。 | |
