| Assay Method Information | |
| | Inhibitory Activity on mTOR Kinase |
| Description: | The model for screening the inhibitory activity of the molecules on mTOR was developed with the LANTHASCREEN™ technology (Lifetechnologies). The reaction substrate (400 nM final), the serial dilutions of the molecules (1% DMSO final) and the enzyme (<1 nM) are successively added to a 384-well plate (Corning 4514) in a final volume of 10 μL per well. After 1 hour of reaction at room temperature, 10 μL of a solution containing 10 mM final of EDTA and 2 nM final of terbium-labeled antibodies are added. After at least 30 minutes of incubation at room temperature, the TR-FRET signal is measured with a suitable microplate reader according to the supplier's recommendations. The data are normalized with positive controls (POS containing a saturating concentration of reference inhibitor) and negative controls (NEG containing 1% DMSO): % inhibition=((X-NEG)*100)/(POS-NEG). The IC50 values are calculated using a 4-parameter logistic model with the aid of the XLFit software (IDBS). |
| Affinity data for this assay | |
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