Phospho-PRAS40 (T246), Human/Mouse, BioAssay(TM) Cell-Based ELISA (AKT1S1, Lobe)

PRAS40 (40kD proline-rich Akt1 substrate), also known as AKT1S1, is a widely expressed component of the cytoplasmic mTOR complex (mTORC1). In response to insulin stimulation, Akt1 phosphorylation of PRAS40 relieves the basal PRAS40-mediated inhibition of mTORC1. Activated mTORC1 is a key effector of the cell survival pathway. PRAS40 phosphorylation by Akt3 or Pim1 similarly exerts an anti-apoptotic effect during tumor progression and neuronal ischemia. Alternate splicing generates an isoform that lacks the N-terminal 130 amino acids.\n\nSample Type:\nWhole cells\n\nIntended Use:\nAn ELISA-based assay using fluorogenic substrates to measure phosphorylated PRAS40 in whole cells.\n\nTest Principle:\nThis BioAssay(TM) Cell-Based ELISA contains the components required to run an ELISA using fluorogenic substrates to measure phosphorylated PRAS40 (T246) in whole cells. This simple and efficient assay eliminates the need to prepare cell lysates and can be used to investigate signaling pathways and the effects of inhibitors on cells. Cells are grown in 96-well plates and stimulated with ligands. Following stimulation, cells are fixed and permeabilized in the wells. The target protein phosphorylation is measured using a double immunoenzymatic labeling procedure. The cells are simultaneously incubated with two primary antibodies: a phospho- specific antibody and a normalization antibody that recognizes the total protein regardless of phosphorylation status. The primary antibodies are derived from different species. Two secondary antibodies recognizing the different species are labeled with either horseradish- peroxidase (HRP) or alkaline phosphatase (AP), and two spectrally distinct fluorogenic substrates for either HRP or AP are used for detection. The fluorescence of the phosphorylated protein is normalized to that of the total protein in each well for the correction of well-to-well variations. This two-wavelength assay results in precise analysis of protein phosphorylation with good reproducibility.\n\nKit Components:\nPhospho-PRAS40 (T246) Antibody, 1x vial\nTotal PRAS40 Antibody, 1x vial\nHRP-conjugated anti-rabbit IgG, 1x110ul\nAP-conjugated anti-mouse IgG, 1x110ul\nSubstrate F1 Concentrate, 1x50ul\nF1 Diluent, 1x10ml\nSubstrate F2, 1x10ml\nBlocking Buffer, 1x35ml\nWash Buffer, 5X, 1x60ml\nMicroplate, 1x96wells\nPlate Sealers, 4x adhesive strips\n\nStorage and Stability:\nStore powder at 4 degrees C liquid at -20 degrees C. Store other components at 4 degrees C. Stable for at least 6 months For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.

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