Detection of mTOR/Raptor activity using a 96 well ELISA based assay Free

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We have developed a convenient and sensitive assay for the measurement of mTOR activity. A GST fusion of the classical mTOR substrate p70S6K is pre-bound to a microtiter plate coated with glutathione. mTOR-containing sample is then added to the sample well in the presence of ATP and manganese containing assay buffer. Active mTOR phosphorylates p70S6K at Thr³⁸⁹. The phosphorylated substrate is detected with a phosphospecific anti-p70S6K(T389) antibody, followed by detection with HRP-antibody conjugate and relative activity is determined by reading absorbance. Using the assay we are able to measure kinase activity of a partially purified mTOR standard generated from an enriched rat brain fraction. The enriched mTOR standard contains both mTOR/Raptor and mTOR/Rictor complexes. The detected mTOR activity is inhibited by the general PI-3K inhibitor wortmannin as well as rapamycin/FKBP12 (IC₅₀ of 8 nM), a specific inhibitor of the mTOR/Raptor complex. The assay was miniaturized to a well volume of 20ul using a 384 well plate format and a fluorescent HRP substrate to improve sensitivity and dynamics. Additionally we detect mTOR activity associated with various mTOR complexes immunoprecipitated from crude 293 cell lysates. Unlike the mTOR /Rictor complex, immunoprecipitation of active mTOR/Raptor requires the use of a CHAPS containing lysis buffer to ensure capture of the intact complex. Phosphorylation of Ser⁴⁷³ on Akt was used to analyze the kinase activity associated with the mTOR/Rictor complex. The described non-radioactive assays are useful for in vitro mTOR inhibitor screening and for assessing the regulation of mTOR in cell signaling.