| Assay Method Information | |
| | FAAH Assay |
| Description: | Frozen (−80 °C) brains (minus cerebella) from adult Wistar or Sprague-Dawley rats were thawed and homogenized in 20 mM HEPES, 1 mM MgCl2, pH 7.0. and thereafter centrifuged at ~35000 × g for 20 min at 4°C. Homogenates were washed (by centrifugation at ~35000 × g for 20 min at 4°C followed by resuspension in buffer) twice and incubated at 37°C for 15 min in order to hydrolyse all endogenous FAAH substrates. After a further centrifugation, the pellets were resuspended in 50 mMTris-HCl buffer, pH 7.4, containing 1 mM EDTA and 3 mM MgCl2 and frozen at −80°C in aliquots until used for assay. For FAAH assay [Boldrup et al., J. Biochem. Biophys. Methods, 60:171-177] test compounds, homogenates (0.5-0.8 μg protein per assay, diluted with 10 mMTris-HCl, 1 mMEDTA pH 7.4) and 25 μL of [3H]AEA in 10 mMTris- HCl, 1 mMEDTA, pH 7.4, containing 1% w/v fatty acid-free bovine serum albumin, final substrate concentration of 0.5 μM) were incubated for 10 min at 37°C (final assay volume 200 μL). Reactions were stopped by placing the tubes on ice. Final assay concentrations of the solvents used for the compounds (ethanol or DMSO) were in the range 1-5%. Activated charcoal (80 μL + 320 μL 0.5 M HCl) was added and the samples were mixed and left at room temperature for about 30 min. Following centrifugation at 2500 rpm for 10 min, aliquots (200 μL) of the supernatants were analyzed for tritium content by liquid scintillation spectroscopy with quench correction. Blank values were obtained by the use of buffer rather than homogenate. |
| Affinity data for this assay | |
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