| Assay Method Information | |
| | Biochemical Assay using Reaction Biology's ADP-Glo Assay |
| Description: | Step 1: Lipid kinases: Reaction Biology Corporation (RBC) currently offers lipid kinases in ADP-Glo formatStep 2: Assay Description Assay principle: The kinase reactions utilize ATP and produce ADP as a byproduct. The ADP production is quantified by ADP-Glo luminescence detection. This is a 3-step reaction: First, the kinase reaction with lipid substrate is carried out in the presence of ATP, and the reaction is quenched and depleted remaining ATP with ADP-Glo™ reagent, and then finally ADP is converted to ATP which is measured using a luciferase/luciferin reaction.Step 3: Assay Procedure: 1. Prepare kinase in freshly prepared Reaction Buffer, kinase without substrate is delivered to background wells; 2. Deliver substrate into the kinase solution and gently mix, deliver to assay wells; 3. Deliver compounds in 100% DMSO into the kinase reaction mixture by Acoustic technology (Echo550; nanoliter range), incubate for 20 min at room temperature; 4. Deliver ATP into the reaction mixture to initiate the reaction; 5. Incubate for 60 min at 30° C.; 6. Quench the reaction with ADP-Glo reagent and incubate for 40 min; 7. Add Detection Mixture and incubate for 30 min; 8. Measure luminescence.Step 4: Data Analysis: The luminescence is converted into M ADP production based on ADP standard curves. The nonlinear regression to obtain the standard curve and IC50 values are performed using Graphpad Prism software. |
| Affinity data for this assay | |
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