| Assay Method Information | |
| | Inhibition Assay |
| Description: | The in vitro inhibitory activity of compounds against human MOGAT-2 is evaluated in this assay. MOGAT-2 transfers an oleoyl group to monooleoyl-glycerol (MAG) from oleoyl-CoA to form dioleoyl-glycerol (DAG) in the intestinal triglyceride resynthesis pathway. The assay takes advantage of Microscint E extraction, which extracts hydrophobic molecules selectively over hydrophilic ones to separate the 14C-oleoyl-CoA from 14C-DAG.Genetically engineered insect SF9 cells express human MOGAT-2. Prepare the cell lysate in 20 mM of NaCl with protease inhibitor (Roche Cat#11873580001). Homogenize the SF9 cells expressing human MOGAT-2 at 15,000 rpm for 20x2 seconds (PT-3100 Polytrone). Centrifuge the homogenate at 1000 g for 10 minutes at 4 C. Collect the supernatant into a separate tube for protein quantification and activity testing. Purify the glycerol monooleate substrate (Spectrum Chemical, CAS#25496-72-4) chromatographically. |
| Affinity data for this assay | |
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