Epigenase JMJD2 Demethylase Activity/Inhibition Assay Kit (Fluorometric)

The Epigenase™ JMJD2 Demethylase Activity/Inhibition Assay Kit (Fluorometric) is a complete set of optimized reagents, designed for an easy and fast fluorometric measurement of JMJD2 activity or inhibition. The antibody-based, immunospecific method directly detects JMJD2-converted demethylated products, rather than by- products, in a 96 stripwell microplate format. The kit has the following advantages:

• 3 hour fluorometric procedure in a 96 stripwell microplate format allows for either manual or high throughput analysis.
• Directly measures JMJD2 activity via a straightforward detection of JMJD2-converted demethylated products, rather than by-products, thus eliminating assay interference caused by thiol-containing chemicals such as DTT, GSH and 2-mercaptoethanol, or caused by detergents/ions such as tween-20, SDS, triton X-100, Fe, and Na.
• Both cell/tissue extracts and purified JMJD2 can be used, which allows for the detection of inhibitory effects of JMJD2 inhibitors in vivo and in vitro.
• Sensitivity is up to 2,000 times higher than formaldehyde release-based JMJD2 assays, allowing activity to be fluorometrically detected from as low as 5 ng of purified JMJD2 enzyme.
• Demethylated H3-K9 standard is included, allowing specific activity of JMJD2 to be quantified. 
• Accurate, reliable, and consistent with extremely low background signals.

About Histone Demethylation
Histone 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 JMJD2. JMJD2 demethylases are found to have potential oncogenic functions. Detection of activity and inhibition of JMJD2 would be important in elucidating mechanisms of epigenetic regulation of gene activation and silencing, and benefit cancer diagnostics and therapeutics.

Principle & Procedure
In this assay, a tri-methylated histone H3-K9 substrate is stably coated onto microplate wells. Active JMJD2s bind to the substrate and remove methyl groups from the substrate. The JMJD2-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 fluorsecence in a fluorescent microplate spectrophometer at 530 excitation and 590 emission. The activity of the JMJD2 enzyme is in turn proportional to the fluorescent intensity measured.