| Assay Method Information | |
| | Measurement of Inhibitory Effect Against DGAT2 Enzyme Activity |
| Description: | In vitro DGAT2 analysis was perfomed used a Phospholipid Flast Pate (Perkin Elmer) based on the principle of SPA (Scintillation Proximity Assay). First, DGAT2 inhibition compounds serially diluted 5 times from 3 nM to 10 nM (final conceentration, 1% DMSO) were mixed i a buffer solution containing 2 ug DGAT2-membrane protein and 20 mM HEPES, 20mM MgCL2, 1 mg/ml BSA, 50 uM 1.2 sn-oleoyl glycerol (Sigma), put in a 96-well flash plate (FlashPlate) and reacted at 37C for 20 minutes, and then 1 uM [14C] ole oil CoA (PerkinElmer, NEC651050UC) was added to be a final volume of 100 uL and further reacted at 27C for 15 minutes. After the enzymatic reaction was completed, 100 uL of isopropanol was added, the plate was sealed with a film, and the plate was the plate was shaken slowly in a plate shaker. The next day, the amplified scintillation signal (cpm) in Topocounter (Packard) was measured to measure the degree of production of [14C]-labeled tracyl glycerol (TG) as a reaction product. The measured value when the compound was not treated was used as a positive control, and the measured value of the compound treated group was calcuated as a relative % to measure the inhibition effect of the compound on TG production. |
| Affinity data for this assay | |
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