Simple two-step protocol with< 1 hour time to answerColorimetric readout at 405 nm compatible with most plate readersResistant to detergent interference common to antibody-based assaysEliminates need for radioactivity, extractions, and/or chromatographySuitable for high throughput analysis
Useful for assaying lysates, immunoprecipitates or inhibitor screening using the nuclear extract provided.-Includes HeLa nuclear extract, a rich source of HDACs 1 & 2 for use as a positive control or as a source of HDAC activity for screening.Compatible with class I & IIb HDAC and sirtuins (with addition of NAD+).The COLOR DE LYS system (Colorimetric Histone de Acetylase Lysyl Substrate/Developer) is a sensitive and convenient alternative to protocols utilizing radiolabeled, acetylated histones or peptide/HPLC methods for the assay of histone deacetylases. The assay procedure has two steps. First, the COLOR DE LYS substrate which comprises an acetylated lysine side chain, is incubated with a sample containing HDAC activity (HeLa nuclear or other extract, purified enzyme, bead bound immunocomplex, etc.). Deacetylation of the substrate sensitizes the substrate so that, in the second step, mixing with the COLOR DE LYS developer causes an increase in yellow color intensity, and absorption at 405 nm.Note: The COLOR DE LYS substrate is efficiently deacetylated by HDAC1 and HDAC2, the major contributors to HDAC activity in HeLa nuclear extracts. It is however, a poor substrate for HDAC3 and recombinant human HDAC8 (BIOMOL International, unpublished results). The activities of other HDAC isotypes with the COLOR DE LYS substrate have yet to be investigated. HDAC3 and HDAC8 do deacetylate the FLUOR DE LYS substrate, which is the basis for the HDAC fluorometric activity assay kit.
Colorimetric detection, HTS
Nuclear Extracts from HeLa Cells (human cervical cancer cell line) (500 µl)Storage: avoid freeze/thaw cycles!COLOR DE LYS Substrate (50 µl; 50mM in DMSO)Storage: -70°CCOLOR DE LYS Developer Concentrate (20x) (100 µl; 20x stock solution, dilute in HDAC assay buffer before use)Storage: -70°C Trichostatin A (HDAC Inhibitor) (100 µl; 0.2mM in DMSO)Storage: -70°CCOLOR DE LYS Deacetylated Standard (30 µl; 10mM in DMSO)Storage: -70°CHDAC Assay Buffer (50mM TRIS-HCl, pH 8.0, 137mM sodium chloride, 2.7 mM potassium chloride, 1mM magnesium chloride) (20 ml)Storage: -70°C1/2 volume microplateStorage: Room temperature