| Assay Method Information | |
| | Fluorogenic Assay |
| Description: | h-FAAH: The fluorescent assay to measure FAAH activity was performed in 96 wells black plates: 2.5 μg of human FAAH-1 membrane preparation were pre-incubated for 50 min at 37° C., in 190 μL of assay buffer (50 mM TrisHCl pH 7.4, 0.05% Fatty acid-free BSA) with 5 μL of inhibitor or 5 μL DMSO to measure FAAH total activity. The background (no activity) samples were prepared using 190 μL of assay buffer without human FAAH-1 and 5 μL of DMSO. The reaction was then started by the addition of 5 μL of substrate (AMC arachidonoyl amide, Sigma) dissolved in DMSO and used at a final concentration of 800 nM. The reaction was carried out for 45 minutes at 37° C. and fluorescence was measured with EnVision 2014 Multilabel Reader (PerkinElmer, Massachusetts, USA) (excitation wavelength 355 nm/emission wavelength 460 nm). The concentration causing half-maximal inhibition (IC50) was determined by nonlinear regression analysis of the Log [concentration]/response curves generated with mean replicate values using a four-parameter Hill equation curve fitting with GraphPad Prism 5 (GraphPad Software Inc., CA, USA). |
| Affinity data for this assay | |
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