The Epigenase™ JARID Demethylase Activity/Inhibition Assay Kit (Fluorometric) is a complete set of optimized reagents, designed for an easy and fast fluorometric measurement of JARID activity or inhibition. The antibody-based, immunospecific method directly detects JARID-converted demethylated products, rather than by- products, in a 96 stripwell microplate format. The kit has the following advantages:
About Histone DemethylationHistone demethylation is the removal of methyl groups in modified histone proteins via histone demethylases. The discovery of histone demethylases demonstrates that histone methylation is not a permanent modification, but rather a more dynamic process. One of the most studied families of histone demethylating enzymes is currently Jumonji domain-containing (JmjC) histone demethylase, which includes JARID. JARIDs function as transcription repressors and might participate in different biological processes through recruitment to different chromosomal regions and differing enzymatic activities. Detection of activity and inhibition of JARID would be important in elucidating mechanisms of epigenetic regulation of gene activation and silencing, and benefit cancer diagnostics and therapeutics.
Principle & ProcedureIn this assay, a tri-methylated histone H3-K4 substrate is stably coated onto microplate wells. Active JARIDs bind to the substrate and remove methyl groups from the substrate. The JARID-demethylated products can be recognized with a specific antibody. The ratio or amount of demethylated products, which is proportional to enzyme activity, can then be fluorometrically measured by reading the fluorescence in a fluorescent microplate spectrophometer at 530 excitation and 590 emission. The activity of the JARID enzyme is in turn proportional to the fluorescent intensity measured.