Figure 1. Treatment of HeLa cells with insulin stimulates phosphorylation of PRAS40 at Thr246, detected by the PathScan® Phospho-PRAS40 (Thr246) Sandwich ELISA Kit #7327, 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.Learn more about how we get our images
|Product Includes||Volume||Solution Color|
|PRAS40 Rabbit mAb Coated Microwells||96 tests|
The PathScan® Total PRAS40 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of PRAS40. A PRAS40 rabbit antibody has been coated onto the microwells. After incubation with cell lysates, PRAS40 (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a PRAS40 mouse detection antibody is added to detect captured PRAS40 protein. Anti-mouse IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of total PRAS40 protein.
Antibodies in kit are custom formulations specific to kit.
PathScan® Total PRAS40 Sandwich ELISA Kit #7331 detects endogenous levels of PRAS40 protein 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.
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 proteins (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).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. PathScan is a trademark of Cell Signaling Technology, Inc.
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