| Assay Method Information | |
| | VPS34 ADP-Glo kinase assay |
| Description: | [0194] Experimental compound in DMSO was dispensed to a 384-well microplate via acoustic dispenser at 50 nl per well. At 2.5 µl per well, an enzyme mix with VPS34 (Carnabio 11-122) at 0.3 nM was pre-incubated with compound for 20 min, then 2.5 µl substrate mix added to initiate reaction with PI:PS (Signalchem P425-59B-1000) at 50 µM, and ATP at 70 µM (ATP Km = 69 µM). Assay buffer conditions were as follows: 50 mM HEPES, pH 7.5, 3 mM MnCl2, 0.01% BSA, 0.01% Brij 35, 1 mM TCEP. The 5 µl reaction was incubated for 90 minutes at room temperature, and then quenched with equal volume of ADP-Glo Reagent (5 µl; Promega). Quenched reaction was incubated for 60 min, followed by addition of an equal volume of Kinase Detection Reagent (10 µl; Promega). Luminescence signal was detected on an Envision (Perkin Elmer). |
| Affinity data for this assay | |
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