Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-PRAS40 (Thr246) Sandwich ELISA Kit #7327

When ordering five or more kits, please contact us for processing time and pricing at sales@cellsignal.com.

Kit Includes Volume Solution Color
PRAS40 Rabbit Ab Coated MIcrowells 96 tests
Biotinylated Phospho-PRAS40(Thr246) Rabbit Detection Ab 11 ml Green
HRP-linked Streptavidin 11 ml Red
TMB Substrate #7004 11 ml
STOP Solution #7002 11 ml
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) #9803 15 ml Yellowish

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

Species Cross-Reactivity

H M

Reactivity Key:  H=Human  M=Mouse
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

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.

Specificity / Sensitivity

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.

ELISA

ELISA

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.

ELISA - Western correlation

ELISA - Western correlation

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.

Background

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).

  1. Cantley, L.C. (2002) Science 296, 1655-7.
  2. Kovacina, K.S. et al. (2003) J Biol Chem 278, 10189-94.
  3. Vander Haar, E. et al. (2007) Nat Cell Biol 9, 316-23.
  4. Sancak, Y. et al. (2007) Mol Cell 25, 903-15.
  5. Oshiro, N. et al. (2007) J Biol Chem 282, 20329-39.

Application References

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