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:
- 3 hour fluorometric procedure in a 96 stripwell microplate format allows for either manual or high throughput analysis.
- Directly measures JARID activity via a straightforward detection of JARID-converted demethylated products, rather than by-products, thus eliminating assay interference caused by thiol-containing chemicals such as DTT, GSH and 2-mercaptoethanol, caused by detergents/ions such as tween-20, SDS, triton X-100, Fe, and Na.
- Both cell/tissue extracts and purified JARID proteins (including JARID1A, JARID1B, JARID1C, and JARID1D) can be used, which allows for the detection of inhibitory effects of JARID inhibitors in vivo and in vitro.
- Sensitivity is up to 1,000 times higher than formaldehyde release-based JARID assays, allowing activity to be fluorometrically detected from as low as 10 ng of purified JARID enzyme.
- Demethylated H3-K4 standard is included, allowing specific activity of JARID 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 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 & Procedure
In 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.
Fig. 1. Schematic procedure for the Epigenase™ JARID Demethylase Activity/Inhibition Assay Kit (Fluorometric).
Fig. 2. Demonstration of high sensitivity of the JARID activity assay achieved by using A549 nuclear extracts with the Epigenase™ JARID Demethylase Activity/Inhibition Assay Kit (Fluorometric).