Abcam’s mTOR pSer2448 in vitro ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for the measurement of Ser2448 of mTOR protein in cell and tissue lysates.
The assay employs an antibody specific to mTOR protein coated onto well plate strips. Samples are pipetted into the wells and mTOR present in the sample is bound to the wells by the immobilized antibody. The wells are washed and an anti-mTOR phospho Ser2448 detector antibody is added. After washing away unbound detector antibody, HRP-conjugated label specific for the detector antibody is pipetted into the wells. The wells are again washed, a TMB substrate solution is added to the wells and blue color develops in proportion to the amount of phosphorylated Ser2448 bound mTOR. The developing blue color is measured at 600 nm. Optionally the reaction can be stopped after 25 – 30 minutes of incubation by adding hydrochloric acid which changes the color from blue to yellow and the intensity can be measured at 450 nm.
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Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase part of two distinct signaling complexes, mTORC1 and mTORC2. These two complexes share four proteins (mTOR, mLST8, DEPTOR, Tti1/tel2), with only mTORC1 containing Raptor and PRAS40 and mTORC2 containing Rictor, mSin1 and Protor1/2. The complex mTORC1 (rapamycin sensitive complex) coordinates inputs from growth factors, stress, energy status, oxygen and amino acids levels to control processes such as protein and lipid synthesis and autophagy. The complex mTORC2 is insensitive to nutrients and rapamycin, but it responds to insulin signaling. It also controls ion transport and cell shape by targeting serum/glucocorticoid protein kinase (SGK1) and protein kinase (PKC-α) respectively.
The canonical regulation of mTORC1 occurs through the TSC/Rheb pathway which receives signals from AKT, AMPK and IKKβ to activate the complex. Phosphorylation of mTOR at Ser2448 is carried out directly by AKT kinase as well as p70S6 kinase acting as a feedback signal. Phosphorylation at this site is a biomarker for the activation state of the PI-3 kinase pathway as well as the activation status of mTOR. Activation of mTOR leads to phosphorylation of PRAS40, raptor and DEPTOR and the consequential activation of mTORC1. Deregulated signaling of mTOR has been implicated in diseases such as cancer, metabolic syndrome, neurodegeneration and aging. Constitutive activation of PI3K-mTORC1 signaling in cancer cells inhibits autophagy, deregulates protein synthesis via 4E-BP1/eIF4E and increases de novo lipid synthesis via SREBP1. Similarly mTOR signaling is a key factor in the regulation of tissue metabolism in the normal and nutrient overload state affecting the hypothalamus, adipose tissue, the liver, skeletal muscle and pancreas.
Kinase subunit of both mTORC1 and mTORC2, which regulates cell growth and survival in response to nutrient and hormonal signals. mTORC1 is activated in response to growth factors or amino-acids. Growth factor-stimulated mTORC1 activation involves AKT1-mediated phosphorylation of TSC1-TSC2, which leads to the activation of the RHEB GTPase that potently activates the protein kinase activity of mTORC1. Amino-acid-signaling to mTORC1 requires its relocalization to the lysosomes mediated by the Ragulator complex and the Rag GTPases. Activated mTORC1 up-regulates protein synthesis by phosphorylating key regulators of mRNA translation and ribosome synthesis. mTORC1 phosphorylates EIF4EBP1 and releases it from inhibiting the elongation initiation factor 4E (eiF4E). mTORC1 phosphorylates and activates S6K1 at 'Thr-421', which then promotes protein synthesis by phosphorylating PDCD4 and targeting it for degradation. Phosphorylates MAF1 leading to attenuation of its RNA polymerase III-repressive function. mTORC2 is also activated by growth. factors, but seems to be nutrient-insensitive. mTORC2 seems to function upstream of Rho GTPases to regulate the actin cytoskeleton, probably by activating one or more Rho-type guanine nucleotide exchange factors. mTORC2 promotes the serum-induced formation of stress-fibers or F-actin. mTORC2 plays a critical role in AKT1 'Ser-473' phosphorylation, which may facilitate the phosphorylation of the activation loop of AKT1 on 'Thr-308' by PDK1 which is a prerequisite for full activation. mTORC2 regulates the phosphorylation of SGK1 at 'Ser-422'. mTORC2 also modulates the phosphorylation of PRKCA on 'Ser-657'.
Expressed in numerous tissues, with highest levels in testis.
Autophosphorylated; when part of mTORC1 or mTORC2.
Endoplasmic reticulum membrane. Golgi apparatus membrane. Mitochondrion outer membrane. Lysosome. Cytoplasm. Nucleus > PML body. Shuttles between cytoplasm and nucleus. Accumulates in the nucleus in response to hypoxia (By similarity). Targeting to lysosomes depends on amino acid availability and RRAGA and RRAGB.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use at an assay dependent concentration.
Dynamic Range of the assay.
The extract was prepared with HeLa cells treated with 50nM of calyculin for 15 minutes. Data was plotted on an XY graph in the log scale and a four parameter algorithm formula was used for curve fitting.
The mTOR pSer2448 ELISA specifically measures the phosphorylated protein.
Calyculin treated HeLa extracts were left untreated (control) or treated with 1:100 dilution of ? Ppase at 34°C. Samples were loaded at 500, 125 and 30 µg/mL on the plate and measured following the kit’s protocol. Treatment of calyculin treated HeLa extracts with ? Ppase decreases the signal by 12 fold at the highest loading.
The mTOR pSer2448 ELISA cross reacts with mouse samples.
NIH3T3 cells were treated with 50 ng/mL of PDGF recombinant protein after serum starvation or BSA control for 30 minutes at 37°C. A protein titration of PDGF treated cells was loaded in triplicate (left panel) alongside 4 replicates of both BSA and PDGF treated cells at 500 µg/mL. BSA and PDGF samples were interpolated from the standard curve and results are shown on the right panel. PDGF increases the levels of phosphorylation at Ser2448 by 2 fold.
Western blot using capture anti-mTOR and detector anti-mTOR pSer2448.
The detector antibody used in this kit specifically detects the phosphorylated mTOR as determined by Western blotting. Samples were loaded on a 8% polyacrylamide gel as follows: (1) Marker (2) calyculin treated HeLa lysate, (3) calyculin treated HeLa lysate dephosphorylated with 1:100 dilution of ? Ppase at 34°C, (4) Hek293T cell lysate. Samples were then diluted in SDS-PAGE buffer and loaded at 40 µg/well. Membranes were blocked with 20% blocking buffer (ab126587) in TBST for 1 hour and incubated with either the detector antibody mTOR pSer2448 or the capture antibody against total mTOR in 10% blocking buffer (ab126587) in TBST overnight. Labeling was carried out with secondary antibodies conjugated to HRP. Treatment with calyculin in HeLa cells induces phosphorylation at Ser2448 and ? Ppase completely dephosphorylates mTOR.
Cui N et al. Role of Triggering Receptor Expressed on Myeloid Cell-1 Expression in Mammalian Target of Rapamycin Modulation of CD8(+) T-cell Differentiation during the Immune Response to Invasive Pulmonary Aspergillosis. Chin Med J (Engl)130:1211-1217 (2017).
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Fu L et al. Lactic acid bacteria-specific induction of CD4(+)Foxp3(+)T cells ameliorates shrimp tropomyosin-induced allergic response in mice via suppression of mTOR signaling. Sci Rep7:1987 (2017).
Read more (PubMed: 28512288) »