| Assay Method Information | |
| | HDAC Enzymatic Assay |
| Description: | Briefly, this fluorogenic assays uses an acetylated lysine tripeptide substrate, amide-linked to a fluorescently quenched aminocoumarin (AMC). Enzyme reactions were run in 50 mM HEPES, 100 mM KCl, 0.001% (v/v) Tween-20, 0.05% (w/v) BSA, pH 7.4. The appropriate substrate concentration for each HDAC was determined using their corresponding Km values. Km values were determined by monitoring the increase of fluorescence of each HDAC at varying substrate concentrations. The increase in fluorescence units was plotted versus substrate concentration. Assays corresponding to HDACs 4, 5, 7, 8, 9 involved a trifluoroacetylated lysine substrate as described in Bradner et al, while HDACs 1, 2, 3, and 6 used an acetyl-Leu-Gly-acetyl-Lys tripeptide as the substrate. Reactions were carriedout in 384-well plates and fluorescence was measured using a multi-label plate reader and plate stacker (Envision, Perkin-Elmer). |
| Affinity data for this assay | |
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