| Assay Method Information | |
| | GOAT activity assay |
| Description: | GOAT activity was assessed using a time-resolved fluorescence energy transfer (TR-FRET) assay in a 384-well format. His-tag human GOAT enzyme was in the form of a cell membrane preparation from sf9 cells infected with hGOAT-V5-His baculovirus. Varying concentrations of test compound with final DMSO concentration kept to 0.5% were added to membrane solution. Human GOAT membrane activity was established in a buffer having final concentration 0.25 mg/mL in 50 mM MOPS, pH7.5; 50 mM KCl; 0.1 mg/mL BSA; 50 μM CHAPS; and 2 mM EDTA. Substrate solution consisting of biotinylated ghrelin peptide (final concentration 100 nM), octanoyl coA (final concentration 2 μM) and palmitoyl CoA (final concentration 50 μM) was added to initiate the reaction. Plates were sealed, centrifuged for 1 minute at 2000 rpm, then incubated at 30° C. for 80 minutes with gentle shaking on an Eppendorf mix plate. Reaction termination and detection mix consisting of chicken anti-active ghrelin antibody (final concentration of 10 nM), Europium W1024-labeled streptavidin (final concentration of 4 nM), GOAT anti-chicken Dylight (final concentration of 12.5 nM), and GS[DAP-oc]-FL-amide inhibitor (final concentration of 1 μM) was added before further incubation for 40 minutes at 30° C. The plate was then read on an Envision in HTRF mode with excitation filter UV (TRF) 340 and first emission filter of APC 665 and a second emission filter of Europium 615. HTRF readings were acquired as per instrument defined LANCE-DELFIA protocol with a delay and window times of 50 μs for both; number of sequential windows: 1; time between flashes: 2000 μs between each of 100 flashes and 10 flashes for the second detector. The HTRF ratio was calculated directly by the instrument as the ratio of 665 window/615 window. Percent inhibition was calculated as 100−(100×(U−NC)/(PC−NC)) where U was the unknown value HTRF ratio (test compound value), NC was the negative control (100% inhibition value generated from a potent inhibitor), and PC was the positive control (100% activity generated from 0.5% DMSO vehicle). IC50 values were generated in GraphPad Prism (Version 4.03) using non-linear regression curve fit and sigmoidal dose response variable slope analysis. |
| Affinity data for this assay | |
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