HDAC-Glo I/II Assay
The HDAC-Glo I/II Assays and Screening System are single-reagent-addition, homogeneous, luminescent assays that measure the relative activity of histone deacetylase (HDAC) class I and II enzymes from cells, extracts or purified enzyme sources. 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, allowing batch processing of multiwell plates. The HDAC assay is broadly useful for class I and II enzymes. The Trichostatin A, included in the HDAC-Glo I/II Screening Systems or available separately, is a known pan HDAC inhibitor that may be used as a positive control inhibitor. The Trichostatin A is supplied at a concentration of 10mM in DMSO. The HeLa Nuclear Extract, included in the HDAC-Glo I/II Screening Systems or available separately, may be used as a source of histone deacetylase activity. The diluted extract also can be used as an HDAC-Glo I/II Assay chemistry control.