| Assay Method Information | |
| | Human DGKζ Kinase Activity Inhibition Assay |
| Description: | For the assay 50 nl of a 100-fold concentrated solution of the test compound in dimethyl sulfoxide (DMSO, Sigma) was pipetted into either a white 1536-well or a white low-volume 384-well microtiter plate (both Greiner Bio-One, Frickenhausen, Germany). Subsequently, 2 μl of a solution of human DGKζ in aqueous assay buffer [50 mM (3-(N-morpholino)propanesulfonic acid (MOPS, pH 7.4, Sigma-Aldrich), 1 mM dithiothreitol (DTT, Sigma-Aldrich), 100 mM NaCl (Sigma-Aldrich), 10 mM MgCl2 (Sigma-Aldrich), 0.1% (w/v) bovine gamma globulin (BGG, Sigma-Aldrich), 1 μM CaCl2 (Sigma-Aldrich)] were added to the wells, and the mixture was incubated for 15 min at 22 C. to allow pre-binding of the test compounds to the enzyme. The reaction was initiated by the addition of 3 μL of substrate solution [preparation described above; 11.7 μM 1,2-dioleoyl-sn-glycerol (=>final conc. in the 5 μL assay volume is 7 μM), 8.33 mM octyl-β-D-glucopyranoside (=>final conc. in 5 μL assay volume is 5 mM), and 91.67 μM adenosine triphosphate (=>final conc. in 5 μL assay volume is 55 μM) in assay buffer] and the resulting mixture was incubated for a reaction time of 20 min at 22 C. The concentration of DGKζ was adjusted depending of the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, a typical concentration is about 0.1 nM. The reaction was stopped by the addition of 2.5 μL of ADP-Glo-reagent (1 to 1.5 diluted with water) and the resulting mixture was incubated at 22 C. for 1 h to convert the ATP not consumed in the kinase reaction completely to cAMP. Subsequently 2.5 μl of the kinase detection reagent (1.2 fold more concentrated than recommended by the producer) were added, the resulting mixture was incubated at 22 C. for 1 h and then the luminescence measured with a suitable measurement instrument (e.g. Viewlux from Perkin-Elmer). The amount of emitted light was taken as a measure for the amount of ADP generated and thereby for the activity of the DGKζ. |
| Affinity data for this assay | |
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