| Assay Method Information | |
| | HDAC8 Fluorimetric Assay |
| Description: | Activity assays were conducted at 25 °C and contained 500 nM HDAC8 enzyme, 150 μM Ac-Arg-His-Lys(Ac)-Lys(Ac)-aminomethylcoumarin substrate (BML-KI178, Enzo Life Sciences), and 0−10 mM inhibitor in assay buffer [25 mM Tris (pH 8.2), 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl2; 250 μM tris(2-carboxyethyl)phosphine was added for the assay of thiol compound 2] in a final volume of 50 μL. Enzyme was first incubated with inhibitor for 5 min before the reaction was initiated with substrate; the most potent inhibitor, compound 1, was also evaluated after incubation for 30 and 60 min to assess the possibility of time-dependent inhibition. After 30 min, reactions were quenched by adding 100 μM M344 (Sigma-Aldrich) and the appropriate Fluor-de-Lys developer (BMLKI176, Enzo Life Sciences, 50 μL). Fluorescence was measured after 45 min using a Fluoroskan II plate reader (excitation at 355 nm, emission at 460 nm). |
| Affinity data for this assay | |
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