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Assay Method Information |
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| | TR-FRET Tracer Assay |
| Description: | IC50s for mTOR interacting compounds were assessed using the FRAP1/mTOR TR-FRET tracer assay (Invitrogen by Life Technologies). FRAP1/mTOR (PV4753) and LanthaScreen Eu-Anti-GST Antibody (PV5594) (total volume of 14 uL) were added to each well of a ProxiPlate-384 Plus (Perkin-Elmer) 384-well plate. Compounds were serially diluted in DMSO (12-point, 4x dilution factor) and 1 uL of diluted compound was then added to each well and mixed by pipetting using a Biomek FX (Beckman Coulter). 5 uL of mTOR Kinase Tracer 314 (PV6087) was added to each well, mixed and plates were incubated at room temperature for 1 hour. Final concentrations of components are: 6 nM FRAP1/3 nM LanthaScreen Eu-Anti-GST Antibody/50 nM mTOR Kinase Tracer 314/unlabeled compounds, 4.8*10^-6-20 uM. Final assay buffer composition is: 50 mM HEPES (pH 7.5), 50 mM NaCl, 5 mM MgCl2, 1 mM EGTA, 0.01% Pluronic F-127. Plates were measured in plate reader (Perkin Elmer, EnVision) using 340 nm excitation. |
| Affinity data for this assay | |
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