PathScan? Total Bad Sandwich ELISA Kit

• 产品编号：CST-7162C      品牌：CST       原厂货号：7162C
• 产品分类：ELISA及其它免疫分析 > ELISA > ELISA检测试剂盒
• 应用分类：

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描述：

CST生产的 PathScan® Total Bad Sandwich ELISA Kit #7162能够检测内源的Bad 蛋白。TPA处理OVCAR8细胞后，能够明显的诱导Bad在Ser112位点的磷酸化，并用PathScan® Phospho-Bad (Ser112) Sandwich ELISA Kit #7182检测。然而，Bad蛋白的总体水平(磷酸化和非磷酸化)仍然需要PathScan® Total Bad Sandwich ELISA Kit #7162 检测(图 1)。在图3中，通过Western检测用Bad antibody在微孔中扑捉的蛋白，在Bad蛋白对应的位置有一条主要的条带。经过严格的内部测试验证，此抗体能够检测指定物种的蛋白。但是也可能能检测其它物种的同源蛋白。CST生产的PathScan® Total Bad Sandwich ELISA Kit是固相夹心酶联免疫吸附检测试剂盒(ELISA)，它能够检测内源Bad总蛋白。Bad rabbit mAb*包裹到微孔的内表面，然后用细胞裂解液孵育，Bad蛋白(磷酸化和非磷酸化)被抗体捕获。蛋白经过严格的洗涤后，加入Bad mouse mAb* 来检测捕捉到的Bad蛋白。抗鼠IgG, HRP连接抗体#7076*用于识别检测抗体的结合。加入HRP的底物，用 TMB 显色。比例换算成Bad 总蛋白的量。*试剂盒中的抗体是为该试剂盒特制的。Bad是Bcl-2促凋亡家族中的成员，它通过转移 Bax 与 Bcl-2 和 Bcl-xL 结合而促进细胞死亡(1,2)。存活因子如IL-3, 能够通过激活细胞内信号通路，导致Bad在Ser112和Ser136位点发生磷酸化，进而抑制凋亡活性(2)。在这些位点的磷酸化促进Bad与 14-3-3 蛋白的结合，从而阻止了Bad与Bcl-2和Bcl-xl的互作(2)。Akt在Ser136位点磷酸化Bad，而促进细胞的存活(3,4)。Bad在体内和体外都能够被p90RSK(5,6)和线粒体锚定PKA磷酸化Ser112位点（7）。PKA对BH3结构域Ser155位点的磷酸化，在阻止Bad和Bcl-xL的二聚化过程中发挥了重要的作用(8-10)。

1. Yang, E. et al. (1995) Cell 80, 285-291.
2. Zha, J. et al. (1996) Cell 87, 619-628.
3. Datta, S.R. et al. (1997) Cell 91, 231-241.
4. Peso, L. et al. (1997) Science 278, 687-689.
5. Bonni, A. et al. (1999) Science 286, 1358-1362.
6. Tan, Y. et al. (1999) J. Biol. Chem. 274, 34859-34867.
7. Harada, H. et al. (1999) Mol. Cell 3, 413-422.
8. Tan, Y. et al. (2000) J. Biol. Chem. 275, 25865-25869.
9. Lizcano, J. et al. (2000) Biochem. J. 349, 547-557.
10. Datta, S. et al. (2000) Mol. Cell 6, 41-51.

原厂资料：

Specificity / Sensitivity

CST's 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.

Description

CST's 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.

Background

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).

1. Yang, E. et al. (1995) Cell 80, 285-291.
2. Zha, J. et al. (1996) Cell 87, 619-628.
3. Datta, S.R. et al. (1997) Cell 91, 231-241.
4. Peso, L. et al. (1997) Science 278, 687-689.
5. Bonni, A. et al. (1999) Science 286, 1358-1362.
6. Tan, Y. et al. (1999) J. Biol. Chem. 274, 34859-34867.
7. Harada, H. et al. (1999) Mol. Cell 3, 413-422.
8. Tan, Y. et al. (2000) J. Biol. Chem. 275, 25865-25869.
9. Lizcano, J. et al. (2000) Biochem. J. 349, 547-557.
10. Datta, S. et al. (2000) Mol. Cell 6, 41-51.

注意事项：

* 12 8-well modules -Each module is designed to break apart for 8 tests.
**Kit should be stored at 4°C with the exception of 10X Cell Lysis Buffer, which is stored at –20°C (packaged separately).