Useful for inhibitor screening or characterizing enzyme kineticsIncludes optimal substrate selected from a panel of acetylated sites in p53 and histonesSupplied with enough recombinant enzyme for 96 assays (1 x 96-well plate)
A FLUOR DE LYS fluorescent assay system. The HDAC3/NCOR1 Fluorescent Activity Assay/Drug Discovery Kit is a complete assay system designed to measure the lysyl deacetylase activity of the recombinant human HDAC3 included in the kit. The kit is ideal for chemical library screening for candidate inhibitors or activators or kinetic assay of the enzyme under varying conditions. The preparation provided with this kit, a complex of HDAC3 with the NCOR1 Deacetylase Activation Domain (DAD), has over 100-fold greater specific activity than recombinant HDAC3 alone and more closely approximates the fully active in vivo form of the enzyme. Another advantage of the complex for drug discovery purposes is the possibility of identifying small molecule disruptors of the HDAC3-DAD interaction. Thus, in addition to active site binding, there is another potential route to inhibition, one more likely to be highly specific to HDAC3. The FLUOR DE LYS HDAC3/NCOR1 assay is based on the FLUOR DE LYS Substrate and FLUOR DE LYS Developer combination. The assay procedure has two steps. First, the FLUOR DE LYS SIRT1 Substrate, which comprises an acetylated lysine side chain, is incubated with HDAC3/NCOR1. Deacetylation of the substrate sensitizes the substrate so that, in the second step, treatment with the FLUOR DE LYS Developer II produces a fluorophore.
Fluorescence microscopy, HTS
HDAC3/NCOR1 Complex (human, recombinant) 67 µl; 30 ng/µl in 50mM TRIS, pH 8.0, 138mM NaCl, 10% glycerol, 1 mg/ml BSA.Storage: -70°C; avoid freeze/thaw cycles!FLUOR DE LYS−SIRT1, Deacetylase Substrate100 µl; 5mM solution in 50mM TRIS/Cl, pH 8.0, 137mM sodium chloride, 2.7mM potassium chloride, 1mM magnesium chloride.Storage: -70°CFLUOR DE LYS Developer II Concentrate (5x)5 x 250 µl; 5x Stock Solution; dilute in assay buffer before use.Storage: -70°CTrichostatin A (HDAC Inhibitor) 100 µl; 0.2mM in DMSO (dimethylsulfoxide).Storage: -70°CFLUOR DE LYS Deacetylated Standard30µl; 10mM in DMSO (dimethylsulfoxide).Storage: -70°CHDAC Assay Buffer II20 ml; (50 mM TRIS/Cl, pH 8.0, 137mM sodium chloride, 2.7mM potassium chloride, 1mM magnesium chloride, 1mg/ml BSA).Storage: -70°CHDAC Assay Buffer20 ml; (50 mM TRIS/Cl, pH 8.0, 137mM sodium chloride, 2.7mM potassium chloride, 1mM magnesium chloride).Storage: -70°C1/2 volume microplateStorage: Room temperature1/2 volume white NBS microplateStorage: Room temperature