| Assay Method Information | |
| | GHS Ligand Binding Assay |
| Description: | Briefly, direct ligand binding experiments were performed using fluorescence energy transfer with the purified receptor labeled with Alexa Fluor 350 at its N terminus and a ghrelin peptide labeled with FITC (JMV 4946). Titration experimentswere carried out with protein concentrations in the 10 nM protein concentration range and increasing ligand concentrations. Competition experiments were carried out by adding increasing concentrations of the competing compound to a receptor-JMV 4946 mixture (100 nM concentration range). Fluorescence emission spectra were recorded at 20 °C between 400 and 600 nm on a Cary Eclipse spectrofluorimeter (Varian) with an excitation at 346 or 488 nm. Buffer contributions were systematically subtracted. The FRET ratio corresponds to the ratio of theacceptor-emitted fluorescence at 520 nm from excitation attwo different wavelengths, 346 and 488 nm. |
| Affinity data for this assay | |
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