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.
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.
|Product Includes||Volume||Solution Color|
|PRAS40 Rabbit mAb Coated Microwells||96 tests|
|Phospho-PRAS40 (Thr246) (C77D7) Rabbit Detection mAb (Biotinylated)||1 ea||Green (Lyophilized)|
|HRP-Linked Streptavidin (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
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.
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
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.
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).
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