| Assay Method Information | |
| | ADP Glo Kinase Activity Assay |
| Description: | Table 4: ULK1 inhibitor potency was determined using the luminescent ADP Glo Kinase Assay (Promega), a universal ADP detection system that measures kinase activity by quantifying the amount of ADP produced during a kinase reaction. ULK1 kinase reactions are performed in a standard 96-well microplate (20 μL reaction volume) at 25° C. for 40 minutes in the presence or absence of the test compound (12-point dilution series starting at 10 μM). Addition of peptide substrate and ATP initiates the kinase reaction.The standard ADP Glo assay is performed in two steps. In step one, following the kinase reaction, an equal volume of ADP-Glo™ Reagent (20 μL) is added to terminate the kinase reaction and deplete the remaining ATP. In the second step, the Kinase Detection Reagent (10 μL) is added to simultaneously convert ADP to ATP and allow the newly synthesized ATP to be measured using a luciferase/luciferin reaction. Step two of the ADP Glo assay is performed in a white 384-well plate (20 μL final volume). Thirty minutes after the addition of the Detection reagent, light generated from the luciferase reaction is measured (1 second exposure) using a microplate compatible luminometer (CLARIOstar, BMG Labtech).Inhibition of ULK1 kinase activity is determined for each test compound by comparison to DMSO only control wells. Inhibitor IC50 was calculated by plotting the change in luminescence after 30 minutes (response) versus inhibitor concentration (in log molar) then applying the four-parameter variable slope curve fit (GraphPad Prism). |
| Affinity data for this assay | |
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