| Assay Method Information | |
| | Human FAAH 1 Assay |
| Description: | Human FAAH 1 activity was determined by measuring the liberation of the highly fluorescent 7-amino, 4-methyl Coumarin (AMC) generated during hydrolysis of the substrate arachidonoyl 7-amino, 4-methyl coumarin amide (AAMCA) by FAAH. Inhibition of human FAAH 1 activity was determined as a percentage reduction of the fluorescence determined in the absence of compound.The assay was carried out in black-walled, clear bottom, 384-well plates. 27.5 dl of human FAAH 1 protein (in FAAH assay buffer: 50 mM Hepes, 0.01% Triton X-100, 1 mM EDTA, 0.5 mg/ml BSA (fatty-acid-free), pH 8.2) was pre-incubated, at 10 nM, with increasing concentrations of compounds (2.5 μl in 100% DMSO) for 1 hour at room temperature. 2.5 d of DMSO was added for total controls (100% FAAH activity) and 2.5 μl of URB-597, a known inhibitor of FAAH activity, (at a final, saturating, concentration of 10 &M) was used for non-specific controls (0% FAAH activity). 20 d of 7.5 μM AAMCA substrate (in FAAH assay buffer) was then added to all wells and incubated at room temperature for a further 4 hours. Fluorescence was determined at an excitation wavelength of 355 nm and an emission wavelength of 460 nm using a Flexstation plate reader (Molecular Devices, UK). Inhibition of human FAAH 1 activity, by the compounds, was determined as the percentage reduction in relative fluorescence units (RFU) compared to the total controls (in the absence of compound) minus the non-specific controls. IC50 values were determined, from 10-point dose response curves, in XL-Ft using a 4-Parameter Logistic Model (Sigmoidal Dose-Response Model). |
| Affinity data for this assay | |
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