| Assay Method Information | |
| | In Vitro DGK Inhibition Assay |
| Description: | The DGKα (Full Length and Near Full Length) ADP Glo assays were performed using an extruded liposome prep at either 5% DAG or 10% DAG. The Enzymatic reactions were carried out in 50 mM MOPS pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 μM CaCl2, and 1 mM DTT (assay buffer). The lipid substrate concentrations were 1.9 mM PS, either 0.25 mM DAG (5% liposome prep) or 0.5 mM DAG (10% liposome prep), and 2.7 mM PC for the extruded liposome reactions. The reactions were carried out at 150 μM ATP. The enzyme concentrations for the DGKα(Near Full Length) and DGKα(FL) were 5 nM.The compound inhibition studies were carried out as follows: 25 nL droplets of each test compound (11 point, 3-fold dilution series for each compound) solubilized in DMSO were transferred to wells of a white 1536 well plate (Corning 3725). A 5 mL enzyme/substrate (substrate being the 5× or 10× liposome preparation) solution at 2× final reaction concentrations was prepared by combining 5 ml of the extruded liposome preparation with 10 nM (2× final) DGKα(Near Full Length) or DGKα(Full Length) prepared as described below) and incubated at room temperature for 10 minutes. Next, 1 μL of the 2× enzyme/substrate solution was added to wells containing the test compound and reactions were initiated with the addition of 1 μL 300 uM ATP. Substrate only was added to DMSO only wells for background measurements. The reactions were allowed to proceed for 1 hr, after which 2 μL Glo Reagent (Promega V9101) was added and incubated for 40 minutes. Next, 4 μL Kinase Detection Reagent was added and incubated for 30 minutes. Luminescence was recorded using an EnVision microplate reader. The percent inhibition was calculated from the ATP conversion generated by no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The compounds were evaluated at 11 concentrations to determine IC50. |
| Affinity data for this assay | |
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