| Assay Method Information | |
| | DGK ADP-Glo Kinase Assay |
| Description: | The DGKa and DGKz kinase assays were performed using Promega ADP-Glo kit (Promega, Cat #V9102). One microliter test compound was added to a 384-well plate. Reactions were performed in a 5 uL volume by adding 2 uL mixture of DGK enzyme and ATP in assay buffer (50 mM MOPS pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM CaCl2, 0.0025% BSA, and 1 mM DTT). The final concentration of DGKa, DGKz and ATP were 10 nM, 5 nM and 15 uM, respectively. The enzyme solution was incubated with test compound at 25 Figure US20250223301A1-20250710-P00003 for 20 min. For the preparation of the substrate of micelles, 1 volume of a 16.1 mM solution of 1,2-dioleoyl-sn-glycerol in chloroform was slowly evaporated using a nitrogen stream. Subsequently, 22.55 volumes of a 510 mM solution of octyl-b-D-glucopyranoside in 50 mM MOPS buffer (pH 7.5) were added, and the mixture was sonicated in an ultrasonic bath for 20s. Then 35 volumes of 50 mM MOPS buffer (pH7.5) were added to yield a solution of 0.28 mM 1,2 dioleoyl-sn-glycerol and 200 mM octyl-b-D-glucopyranoside, which was aliquoted, flash-frozen in liquid nitrogen, and stored at −80° C. until use. The reaction was initiated by the addition of 2 uL of substrate solution (7 uM 1.2-dioleoyl-sn-glycerol, 5 mM octyl-b-D-glucopyranoside) in the 5 uL assay volume. The resulting mixture was incubated at 25° C. for 40 minutes. Next, 5 uL of ADP-Glo-reagent was added to the plate and incubated for 40 minutes. Then 10 uL of kinase detection reagent was added and incubated for 30 minutes. Luminescence was recorded using an Tecan SPARK microplate reader. 1% DMSO vehicle was used as control and no enzyme well was used as blank well. |
| Affinity data for this assay | |
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