| Assay Method Information | |
| | GOAT Activity Assay |
| Description: | Assays were performed with ~100 μg of membrane protein, as determined by a Bradford assay. The membrane fraction was preincubated with 1 μM methyl arachidonyl fluorophosphonate (MAFP) and inhibitor or vehicle as indicated in 50 mM HEPES (pH 7.0) for 30 min at room temperature [McGovern-Gooch et al., Endocrinology, 157:4330-4338]. Reactions were initiated via the addition of 500 μM octanoyl CoA and 1.5 μM fluorescently labeled ghrelin mimetic, GSSFLCAcrylodan, for a total volume of 50 μL, and were incubated for 3 h at room temperature in the dark. Reactions were stopped with the addition of 50 μL of 20% acetic acid in isopropanol, and solutions were clarified by proteinprecipitation with 16.7 μL of 20% trichloroacetic acid, followed by centrifugation (1000g for 1 min). The supernatant was then analyzed by reverse phase HPLC, as previously described [Darling et al., Anal. Biochem., 437:68-76]. |
| Affinity data for this assay | |
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