| Assay Method Information | |
| | Rat FAAH Inhibition Assay |
| Description: | Active rat FAAH protein (30-579) was isolated as described in the literature. The coding sequence of amino adds 30-579 of rat FAAH were cloned into the expression vector pET28a to provide an N-terminal His-tag. Following expression, the His-tagged FAAH (30-579) was isolated using a method based on Patricelli at al., 1998; Biochemistry vol 37, p 15177 with a combination of chelating sepharose, heparin sepharose and size exclusion chromatography.FAAH 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 FAAH 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 μl of FAAH 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 120 nM, with increasing concentrations of compounds (2.5 μl in 100% DMSO) for 0, 1 or 3 hours at room temperature. 2.5 μl 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 μM) was used for non-specific controls (0% FAAH activity). 20 μl of 7.5 μM AAMCA substrate (in FAAH assay buffer) was then added to all wells and incubated at room temperature for a further 1.5 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 FAAH 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-Fit using a 4-Parameter Logistic Model (Sigmoidal Dose-Response Model). |
| Affinity data for this assay | |
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