| Assay Method Information | |
| | Fluorogenic Assay |
| Description: | h-AC:The assay was run in 96-well microplates (Black OptiPlate-96 F; PerkinElmer, Massachusetts, USA) in a total reaction volume of 100 μL. h-AC protein preparation (2.0 μg) was pre-incubated for 10 min with various concentrations of test compounds or vehicle control (DMSO 5%) in 25 mM sodium acetate buffer (pH 4.5). N-[(1S,2R)-2-hydroxy-1-(hydroxymethyl)-4-(2-oxochromen-7-yl)oxybutyl]dodecanamide was used as substrate (5.0 μM) and the reaction carried for 3 h at 37° C., stopped with MeOH, and treated with NaIO4 (fresh solution in 100 mM glycine/NaOH buffer pH 10.6) followed by 2 h incubation at 37° C. in the dark. 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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