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.The complex for drug discovery purposes is the possibility of identifying small molecule disruptors of the HDAC3-DAD interaction
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.
In addition to active site binding, there is another potential route to inhibition, one more likely to be highly specific to HDAC3.
Delivery information: Kit Contains HDAC3/NCOR1 Complex (human, recombinant) 67 µl; 30 ng/µL in 50mM TRIS, pH 8.0, 138mM NaCl, 10% glycerol, 1 mg/ml BSA, avoid freeze/thaw cycles, Fluor de Lys®−SIRT1, Deacetylase Substrate, (100 µL; 5mM solution in 50mM TRIS/Cl, pH 8.0, 137mM sodium chloride, 2.7mM potassium chloride, 1mM magnesium chloride), Fluor de Lys® Developer II Concentrate (5x) 5 x 250 µL; 5x Stock Solution; dilute in assay buffer before use, Trichostatin A (HDAC Inhibitor) 100 µL; 0.2mM in DMSO (dimethylsulfoxide), Fluor de Lys® Deacetylated Standard 30µL; 10mM in DMSO (dimethylsulfoxide), HDAC Assay Buffer II 20 mL; (50 mM TRIS/Cl, pH 8.0, 137mM sodium chloride, 2.7mM potassium chloride, 1mM magnesium chloride, 1mg/ml BS), HDAC Assay Buffer 20 mL; (50 mM TRIS/Cl, pH 8.0, 137mM sodium chloride, 2.7mM potassium chloride, 1mM magnesium chloride, 1/2 volume microplate and 1/2 volume white NBS microplate.
