| Assay Method Information | |
| | DGKalpha and DGKzeta Biochemical Assays |
| Description: | Compounds of the present invention were prepared into 10 mM DMSO solution and 10 nL of stock was transferred into 384 plates (Optiplate 384 plate) using Echo550. DMSO was used as high control, and ATP substrate buffer was used as a low control. A 1× enzyme assay buffer was prepared (Hepes, pH 7.0 25 mM, BSA 0.05%, Triton-X100 0.002%, CaCl2) 1 μM, MgCl2 10 mM, DTT 2 mM). The enzyme assay was performed by diluting enzyme DGKα (1 μg/μL DGKα, Carna12-101, SEQ ID NO: 3) or DGKζ (1μ/μL DGKζ, Carna 12-110, SEQ ID NO: 4) using 1× assay buffer. OAG (1-oleoyl-2-acetyl-sn-glycerol, 25 mg/ml, Avanti 8001000) and PS (10 mg/ml, Avanti 840032P) were mixed at the ratio of 1:2. A 1× substrate solution was prepared with 1× assay buffer by 100-fold dilution. The substrate solution was sonicated on ice for 1 min. The pure ATP was added to the substrate solution (DGKα:400 μM). 5 μL of the enzyme solution were added to the 384 well plate, and the plate was spun for 1 min at 1000 rpm and incubated for 30 mins at RT. 5 μL of 1× substrate solution were added to the 384 well plate, the plate was spun and then incubated for 45 mins at RT. 10 μL ADP-Glo detergent was added to stop the assay. After 60 mins at RT, 20 μL ADP-Glo Detection buffer was added as the final step. Plate was read after 45 min incubation at RT. |
| Affinity data for this assay | |
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