| Assay Method Information | |
| | DGKA Inhibition ADP-Glo Assay |
| Description: | The DGKA inhibition reactions were performed using ADP-Glo assay. The reactions were carried out in 50 mM HEPES, 100 mM NaCl, 10 mM MgCl2, 1 mM CaCl2, 0.005% TritonX-100 and 1 mM DTT as working solution (pH=7.5). 30 nL DMSO solution of each test compound (Top concentration 0.4 μM with 10 point, 4-fold dilution series for each compound) were transferred to 384 well plate by Echo and 5 μL DGKA (SignalChem, D21-10BG) enzyme working solution at 2×final reaction concentration was added to each well. After incubated at 25° C. for 15 minutes, 5 μL substrate working solution contain 40 μM ATP (Promega, V915B) and 200 μM DLG (SignalChem, D430-59) were added to initiate reaction. After enzymatic reaction at 25° C. for 60 min, ADP Glo assay reagents (Promega, V9102) were added and luminescence was recorded using an EnVision following the instruction of manual. The percent inhibition was calculated from luminescence by no enzyme control reactions for 100% inhibition and DMSO only reactions for 0% inhibition. The IC50 value was calculated via a regression analysis of the inhibition rate. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |