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Assay Method Information

Assay Name:  Enzymatic HDAC Activity Assay
Description:  Biochemical assays of HDAC activity were carried out by Nanosyn in a reaction volume of 10 ul in 384-well microplates. A standard enzymatic reaction contained 5 ul of 2x HDAC inhibitor, 4 ul of 2.5x enzyme, and 1 ul of 10x substrate in assay buffer (100 mM HEPES, pH 7.5, 25 mM KCl, 0.1% BSA, 0.01% Triton X-100, 1% DMSO). Final concentration of all HDACs in the enzymatic assays was between 0.5 and 5 nM. A final substrate concentration of 1 uM FAM-RHKK(Ac)-NH2 or FAM-RHKK(trifluoroacetyl)-NH2 was used in all assays and found to be below the determined Km,app for each enzyme. All inhibitors were serially diluted in DMSO prior to cross-dilution in assay buffer and were incubated with enzyme for 15 min prior to initiating the reaction by the addition of substrate. After incubation for 3 h, the reaction was terminated by the addition of EDTA and SDS to final concentrations of 24 mM and 0.04%, respectively. The product and substrate in each reaction were separated using a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, MA) run on a Caliper LC3000 (Caliper Life Sciences). The separation conditions used a downstream voltage of -800V, an upstream voltage of -3000 V, and a screening pressure of -1.4 p.s.i. The product and substrate fluorescence was excited at 488 nm and detected at 530 nm.
Affinity data for this assay
 

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Last update November 1, 2007
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