| Assay Method Information | |
| | mTOR ATP-Binding Assay |
| Description: | 1. 8-point serial dilutions of compounds (10 mM stock) are performed in 90% DMSO in a 384-well "masterplate" and 50 nL is transferred onto 384-well assay plates (white polystyrene small volume; Matrix/Thermo Scientific Cat. No. #4365). 2. The final volume of the assay is 10 μL and the order of addition is as follows: 50 nL of compounds dilution; 5 μL of a mixture of GST-mTOR and Europium anti-GST antibody with or without the PI3K/mTOR inhibitor P1-103 (3-(4-(4-morpholinyl)pyrido[3',2':4,5]furo[3,2-d]pyrimidin-2-yl)phenol, Calbiochem); 5 μL of tracer-314. Incubated for 60 minutes at room temperature. TR-FRET measured in Biotek Synergy2 reader at: Excitation 340 nm/emission 665 nm; Excitation 340 nm/emission 620 nm. The assay buffer consists of 50 mM HEPES pH 7.5, 5 mM MgCl2, 1 mM EGTA, 0.01% Pluronic F-127. Tracer-314 (Alexa Fluor® 647-labeled ATP-competitive kinase inhibitor; Cat. No. PV6087), Europium anti-GST antibody (Cat. No PV5594) and N-terminally GST-tagged truncated human mTOR (FRAP1) (Cat. No PV4754) are available from Invitrogen. The following final concentrations are used: 3 nM GST-mTOR; 1 nM Europium anti-GST antibody; +/− 10 μM P1-103; and 10 nM tracer-314. The final concentrations of diluted compounds are 9091; 2730; 910; 273; 91; 27; 9; and 3 nM. The final concentration of DMSO is 0.45%. The following controls are used: High signal: solvent vehicle, GST-mTOR, Eu-anti-GST antibody, tracer-314; Low signal: solvent vehicle, GST-mTOR, Eu-anti-GST antibody, P1-103, tracer-314. |
| Affinity data for this assay | |
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