Epigenase JMJD3/UTX Demethylase Activity/Inhibition Assay Kit (Fluorometric)

Product Description

The Epigenase™ JMJD3/UTX Demethylase Activity/Inhibition Assay Kit (Fluorometric) is a complete set of optimized reagents designed for measuring activity/inhibition of JMJD3 and UTX using nuclear extracts or purified enzymes from a broad range of species such as mammals, plants, fungi, and bacteria, in a variety of forms including, but not limited to cultured cells and fresh and frozen tissues. There are currently a limited number of methods used for detecting JMJD3/UTX activity/inhibition. The traditional method is based on the measurement of formaldehyde release, a by-product of JMJD3/UTX enzymatic reaction, and has significant weaknesses: (1) large amounts (at µg level) of substrate and enzyme are required; (2) nuclear extracts from cell/tissues cannot be used; (3) redox-sensitive JMJD3/UTX inhibitiors are not suitable for testing with such methods; (4) high interference by SDS, DMSO, thiol-containing chemicals, and ions, which are often contained in enzyme solutions, tested compound solvents, and assay buffers; and (5) less accurate than direct measurement of JMJD3/UTX-converted demethylated products. The Epigenase™ JMJD3/UTX Demethylase Activity/Inhibition Assay Kit (Fluorometric) addresses these issues with the following advantages:

• 3-hour fluorometric procedure in a 96 stripwell microplate format allows for either manual or high throughput analysis
• Directly measures JMJD3 or UTX activity via a straightforward detection of JMJD3/UTX-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 containing JMJD3/UTX demethylases and purified JMJD3 or UTX proteins can be used, which allows for the detection of inhibitory effects of JMJD3/UTX inhibitors in vivo and in vitro
• Sensitivity is up to 200 times higher than formaldehyde release-based JMJD3/UTX assays, allowing activity to be detected from as low as 10 ng of purified JMJD3 and UTX enzyme
• Demethylated H3-K27 standard is included, allowing specific activity of JMJD3/UTX to be quantified
• Accurate, reliable, and consistent with extremely low background signals

Background Information

Lysine histone methylation is one of the most robust epigenetic marks and is essential for the regulation of multiple cellular processes. The methylation of H3-K27 seems to be of particular significance, as it is associated with repression regions of the genome. H3-K27 methylation was considered irreversible until the identification of a large number of histone demethylases indicated that demethylation events play an important role in histone modification dynamics. So far at least 2 classes of H3-K27 specific histone demethylase, JMJD3 (KDM6B), and UTX (KDM6A) have been identified. The JMJD3 and UTX can remove di- and tri-methylation from H3-K27. JMJD3 and UTX demethylases are JmjC-domain-containing proteins and catalyze the removal of methylation by using a hydroxylation reaction with a required iron and a-ketoglutarate as cofactors.

JMJD3 and UTX demethylases are found to have potential oncogenic functions. For example, JMJD3 is amplified in prostate cancer and UTX mutation was found in multiple cancer types, including kidney cancer and multiple myeloma. Detection of activity and inhibition of JMJD3/UTX would be important in elucidating mechanisms of epigenetic regulation of gene activation and silencing and benefiting cancer diagnostics and therapeutics.

Principle & Procedure

The Epigenase™ JMJD3/UTX Demethylase Activity/Inhibition Assay Kit (Fluorometric) contains all reagents necessary for the measurement of JMJD3/UTX activity/inhibition. In this assay, trimethylated histone H3-K27 substrate is stably coated onto strip wells. Active JMJD3/UTX binds to the substrate and removes methyl groups from the substrate.  The JMJD3/UTX-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 absorbance density (OD) in a microplate reader. The activity of JMJD3/UTX enzyme is proportional to the relative fluoroescent units measured.

Performance Data

Fig.1. Demonstration of high sensitivity of JMJD3/UTX activity assay achieved by using UTX recombinant protein with Epigenase™ JMJD3/UTX Demethylase Activity/Inhibition Assay Kit (Fluorometric).