| Assay Method Information | |
| | mTOR Biochemical Assay |
| Description: | mTOR Kinase reactions were performed in a 10 μL volume in low-volume 384-well plates. Typically, PerkinElmer model 6008260 plates were used. The composition of the 1× kinase reaction buffer was: 50 mM HEPES pH 7.5, 0.01% Tween 20, 1 mM EGTA, 10 mM MnCl2, and 2 mM DTT. The solution of mTOR enzyme (ThermoFisher, #PR8683B; the final mTOR concentration: 0.5 μg/mL) was added, and the compounds were serially diluted to the final maximal concentration of 100 nM (3 fold series dilution, a total of 10 doses). GFP-4E-BP1 (the final concentration: 0.4 μM) and the ATP solution (the final ATP concentration: 3 μM) were added to the 384-well plate. The plate was incubated at 25° C. for 1 hour, and 10 μL of the EDTA solution (20 mM) and Tb-labeled anti-p4E-BP1 antibody (4 nM) in TR-FRET dilution buffer were added to each well. The plate was sealed, incubated 30 min at 25° C., and read on a plate reader configured for LanthaScreen™ TRFRET. |
| Affinity data for this assay | |
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