| 货号 | 7327C |
| 描述 | The PathScan® Phospho-PRAS40 (Thr246) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of PRAS40 when phosphorylated at Thr246. A PRAS40 rabbit antibody has been coated onto the microwells. After incubation with cell lysates, PRAS40 protein (phosphorylated and nonphospho) is captured by the coated antibody. Following extensive washing, a biotinylated phospho-PRAS40 (Thr246) rabbit detection antibody is added to detect the captured phospho-PRAS40 (Thr246) protein. HRP-linked streptavidin 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 PRAS40 phosphorylated at Thr246.PathScan ® Phospho-PRAS40 (Thr246) Sandwich ELISA Kit是一种固相三明治酶联免疫吸附方法（ELISA），用来检测在Thr246位点磷酸化的内源性的PRAS40蛋白。兔源PRAS40抗体已经包被在孔中，与细胞的裂解液共孵育后， PRAS40 (磷酸化和非磷酸化)蛋白被已包被的抗体捕获。经过彻底的洗涤，加入biotinyl-ated phospho-PRAS40 (Thr246) rabbit抗体捕获phospho-PRAS40 (Thr246)蛋白。HRP连接的亲和素链霉素用来识别结合的抗体。加入HRP底物TMB后显色。颜色的吸光度值与Thr246位点磷酸化的内源性的PRAS40蛋白的含量呈正相关。 |
| 反应种属 | Human/Mouse |
| 应用 | ELISA |
| 目标/特异性 | PathScan® Phospho-PRAS40 (Thr246) Sandwich ELISA Kit #7327 detects endogenous levels of PRAS40 protein when phosphorylated at Thr246 as shown in Figure 1. Kit sensitivity is shown in Figure 2. 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 | Many growth factors and hormones induce the phosphoinositide 3-kinase signaling pathway, which results in the activation of downstream effector proteins such as the serine/threonine kinase Akt (1,2). One known Akt substrate is a 40 kDa, proline-rich protein (PRAS40) that binds to 14-3-3 protein (2). PRAS40 also binds mTOR to transduce Akt signals to the mTOR complex. Inhibition of mTOR signaling stimulates PRAS40 binding to mTOR, which in turn inhibits mTOR activity (3). PRAS40 interacts with Raptor in mTOR complex 1 (mTORC1) in insulin-deprived cells and inhibits the activation of the mTORC1 pathway mediated by the cell cycle protein Rheb. Phosphorylation of PRAS40 by Akt at Thr246 relieves PRAS40 inhibition of mTORC1 (4). mTORC1 in turn phosphorylates PRAS40 at Ser183 (5) |
| 存放说明 | 4C and -20C |
| 参考文献 | Cantley, L.C. (2002) Science 296, 1655-7. Kovacina, K.S. et al. (2003) J Biol Chem 278, 10189-94. Vander Haar, E. et al. (2007) Nat Cell Biol 9, 316-23. Sancak, Y. et al. (2007) Mol Cell 25, 903-15. Oshiro, N. et al. (2007) J Biol Chem 282, 20329-39. |
Figure 1. Treatment of HeLa cells with insulin stimulates phosphorylation of PRAS40 at Thr246, detected by the PathScan® Phospho-PRAS40 (Thr246) Sandwich ELISA Kit, but does not affect the levels of total PRAS40 detected by PathScan® Total PRAS40 Sandwich ELISA Kit #7331. HeLa cells (80-90% confluent) were treated with 1.7 μM insulin for 15 minutes and lysed. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using PRAS40 (D23C7) XP® Rabbit mAb #2691 (left panel) or Phospho-PRAS40 (Thr246) (C77D7) Rabbit mAb #2997 (right panel) are shown in the bottom figure. Figure 1，采用胰岛素处理Hela 细胞，使得PRAS40蛋白在Thr246位点磷酸化，采用PathScan ® Phospho-PRAS40 (Thr246) Sandwich ELISA Kit检测。 PathScan ® Total PRAS40 Sandwich ELISA Kit #7331 检测总PRAS40蛋白未发生变化。HeLa采用1.7 μM的胰岛素处理15分钟后裂解。在450nm处读取吸光度值（上图），相对应western blot采用抗体为PRAS40 (D23C7) XP ® Rabbit mAb兔单抗 #2691 (左泳道) 或 Phospho-PRAS40 (Thr246) (C77D7) Rabbit mAb兔单抗 #2997 （右泳道） ，见下图。 | |
Figure 2. The relationship between the protein concentration of lysates from untreated and insulin-treated HeLa cells and the absorbance at 450 nm using the PathScan® Phospho-PRAS40 (Thr246) Sandwich ELISA Kit is shown. Firure2 未处理组和胰岛素处理的Hela细胞提取物中蛋白浓度与450nm处吸光度值的关系图。所用试剂盒为PathScan ® Phospho-PRAS40 (Thr246) Sandwich ELISA Kit 。 |
