The HDAC-Glo™ I/II Assays are single-reagent-addition, homogeneous, luminescent assays that measure the relative activity of histone deacetylase (HDAC) class I and II enzymes using purified enzymes, extracts or cells directly in culture plates. The add-mix-measure protocol allows for simple measurement of deacetylating activities and easy implementation from benchtop to screening. The HDAC-Glo™ I/II Assays can be used to determine HDAC inhibitor potency, selectively profile HDAC inhibitors, correlate HDAC inhibitor potency with cellular fate in same-well multiplexed viability assays or determine off-target HDAC effects of compounds.
The assays use an acetylated, live-cell-permeant, luminogenic peptide substrate that can be deacetylated by HDAC activities. Deacetylation of the peptide aminoluciferin substrate is measured using a coupled enzymatic system in which a protease in the Developer Reagent cleaves the deacetylated peptide from the aminoluciferin substrate, releasing aminoluciferin, which is quantified in a reaction using Ultra-Glo™ recombinant firefly luciferase.
The assay reaction is typically complete within 15–45 minutes with no sample manipulation. The HDAC-mediated luminescent signal is persistent, with a half-life of greater than 3 hours, making the protocol amenable to automation in high-throughput formats and compatible with luminometers without injectors and allowing batch processing of multiwell plates. The HDAC assay is broadly useful for class I and II enzymes. The HDAC-Glo™ I/II Assays are highly sensitive, allowing you to obtain a dynamic range 10- to 100-fold higher than comparable fluorescence methods.