| Assay Method Information | |
| | Biochemical Assay for VPS34 |
| Description: | Activity of VPS34 kinase was determined using an ADP-Glo kinase activity assay (Promega Corp). Assays were conducted in 384-well plates (5 μL assay volume) using 25 nM VPS34 (ThermoFisher Scientific), 100 μg/μL PI:3PS Lipid Kinase Substrate (Promega Corp.), and 1 mM ATP in kinase buffer. Inhibition of VPS34 was measured by adding serial diluted test compound (final assay concentration of 0.1% DMSO) followed by a 1-hour incubation at 37° C. ADP-Glo reagent was then added (5 mL) followed by a 40 min incubation at rt. Kinase detection reagent was then added (10 mL) and followed by a 60 min incubation at rt. Luminescence was then detected. The RLU at each compound concentration of was converted to percent inhibition using controls (i.e., reaction with no test compound and reaction with a known inhibitor) and IC50 values were calculated by fitting a four-parameter sigmoidal curve to the data using GeneData Screener software. |
| Affinity data for this assay | |
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