| Assay Method Information | |
| | Fluorogenic Assay |
| Description: | h-NAAA: The assay was run in 96-well microplates (Black OptiPlate-96 F; PerkinElmer, Massachusetts, USA), in a total reaction volume of 200 μL. h-NAAA protein preparation (4.0 μg) was pre-incubated for 30 min with various concentrations of test compounds or vehicle control (DMSO 5%) in 100 mM citrate/phosphate buffer (pH 4.5) containing 3.0 mM DTT, 0.1% NP40 0.1%, 0.05% BSA, 150 mM NaCl. N-(4-methyl-2-oxo-chromen-7-yl)-hexadecanamide (PAMCA) was used as a substrate (2.0 μM) and the reaction carried for 50 min at 37° C. Fluorescence was measured with EnVision 2014 Multilabel Reader (PerkinElmer, Massachusetts, USA) using an excitation wavelength of 355 nm and emission 460 nm. IC50 values were calculated by non-linear regression analysis of log [concentration]/inhibition curves using GraphPad Prism 5 (GraphPad Software Inc., CA, USA) applying a standard slope curve fitting. |
| Affinity data for this assay | |
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