PerkinElmer, 1744 William St., Montreal, QC H3J 1R4, Canada
Sponsored by PerkinElmer
In this study, we describe the development and optimization of homogeneous antibody-based assays for measuring the catalytic activity of a series of epigenetic lysine-modifying enzymes acting on histone H3 Lys4 (SIRT1 deacetylase and LSD1 demethylase), Lys27 (HDAC1 deacetylase, EZH2 methyltransferase and JMJD3 demethylase) and Lys36 (JMJD2A demethylase). Two different non-radioactive, no-wash technologies were used for detection of the enzymatic reaction products: amplified luminescent proximity homogeneous (AlphaLISA®) assay and time-resolved Förster energy transfer (LANCE® Ultra) assay.
Results demonstrated that all assays were sensitive, rapid and robust (Z’ factors ≥ 0.69), requiring only nanomolar concentrations of enzyme and peptide. Furthermore, profiling of known inhibitors for each epigenetic enzyme showed the expected potency with either technology. These assays will therefore be ideal for the identification of selective small molecule inhibitors. The approach described here is broadly suitable for measuring the catalytic activity of other histonemodifying enzymes by combining the appropriate biotinylated histone-derived peptides and mark-selective antibodies.