| Assay Method Information | |
| | Inhibitory Effect Against DGKalpha Enzyme |
| Description: | First, 3×OAG (3 mM)/ATP (0.45 mM) substrate solution was prepared from 1× substrate assay buffer (40 mM MOPS (pH 7.2), 20 mM MgCl2, 1 mM DTT, 0.4 mM CaCl2), 3 mM sodium deoxycholate, 100 mM NaCl, 0.1 mg/mL BSA, 0.12% NP-40), and vortexed thoroughly for 3 minutes to induce detergent-lipid micelle formation. Then, 3×DGKα (7.5 nM) enzyme solution was prepared from 2× enzyme assay buffer (80 mM MOPS (pH 7.2), 2 mM DTT, 200 mM NaCl, 0.2 mg/mL BSA) and vortexed for a short time. After preparing the above two solutions, a half-area opaque 96-well assay plate was prepared, and 10 μL of 3× diluted compound solution (30 μM to 0 μM) was transferred to each well. Next, 10 μL of 3×DGK enzyme solution was transferred to the same plate, mixed by pipetting, and then 10 μL of 3×OAG/ATP substrate solution was added to the assay plate and mixed well. The plate was incubated at room temperature for 20 minutes for the enzyme reaction. Next, 15 μL of ADP-Glo reagent was added to each well and mixed by pipetting, followed by incubating the plate at room temperature for 40 minutes to deplete the remaining ATPs. After this step, 30 μL of kinase detection reagent was added and mixed, and the plate was incubated at room temperature for an additional 20 minutes and luminescence was measured by Envision to calculate the IC50 value of each compound. |
| Affinity data for this assay | |
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