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


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Suggested Workflow
Nuclear Protein Extraction
Histone Demethylase Assay
Schematic procedure for the Epigenaseā„¢ JMJD3/UTX Demethylase Activity/Inhibition Assay Kit (Colorimetric).
Demonstration of high sensitivity of JMJD3/UTX activity assay achieved by using UTX recombinant protein with Epigenaseā„¢ JMJD3/UTX Demethylase Activity/Inhibition Assay Kit.
Input Type: Nuclear Extracts, Purified Enzyme
Research Area: Histone Methylation
Target Application: Activity Measurement
Vessel Format: 96-Well Plate
100% Guarantee: 6 months
Catalog No.SizePriceQty
P-3084-4848 assays $359.00 
P-3084-9696 assays $579.00 
Availability: Usually Ships In 1-2 Days 
Product Overview

The Epigenase™ JMJD3/UTX Demethylase Activity/Inhibition Assay Kit (Colorimetric) 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 intereference 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 a direct measurement of JMJD3/UTX-converted demethylated products. The Epigenase™ JMJD3/UTX Demethylase Activity/Inhibition Assay Kit addresses these issues, with the following advantages:

  • 3 hour colorimetric 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 (Colorimetric) 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 colorimetrically measured by reading the absorbance density (OD) in a microplate reader. The activity of JMJD3/UTX enzyme is proportional to the OD intensity measured.

Product Components

WB (10X Wash Buffer)
JE1 (JMJD Assay Buffer)
JE2 (JMJD Substrate, 50 µg/ml)*
JE3 (JMJD Assay Standard, 50 µg/ml)*
JE4 (Capture Antibody, 1000 µg/ml*)
JE5 (Detection Antibody, 400 µg/ml)*
DS (Developer Solution)
SS (Stop Solution)
Co-factor 1*
Co-factor 2*
Co-factor 3*
8-Well Assay Strips (With Frame)
Adhesive Covering Film
User Guide

*Spin the solution down to the bottom prior to use. 

User Guide & MSDS

[User Guide]*
*Always use the actual User Guide that shipped with your product. Is the above file locked? You can also request user guides by emailing info@epigentek.com along with your contact information and institution name.

[Safety Data Sheet]
Product Citations

Biswas M et. al. (January 2019). MBD3/NuRD loss participates with KDM6A program to promote DOCK5/8 expression and Rac GTPase activation in human acute myeloid leukemia. FASEB J. :fj201801035R.

Mao J et. al. et. al. (August 2018). Effects of chidamide and its combination with decitabine on proliferation and apoptosis of leukemia cell lines Am J Transl Res. 10(8):2567-2578.

Bai L et. al. (August 2018). Glutaminolysis Epigenetically Regulates Anti-Apoptotic Gene Expression in IPF Fibroblasts. Am J Respir Cell Mol Biol.

Qureshi R et. al. (June 2018). A prokineticin-driven epigenetic switch regulates human epicardial cell stemness and fate. Stem Cells.

Hsu AT et. al. (June 2018). Epigenetic and transcriptional regulation of IL4-induced CCL17 production in human monocytes and murine macrophages. J Biol Chem.

Wulansari N et. al. (October 2017). Vitamin C-Induced Epigenetic Modifications in Donor NSCs Establish Midbrain Marker Expressions Critical for Cell-Based Therapy in Parkinson's Disease. Stem Cell Reports. 9(4):1192-1206.

Mathur R et. al. (February 2017). Inhibition of demethylase KDM6B sensitizes diffuse large B-cell lymphoma to chemotherapeutic drugs. Haematologica. 102(2):373-380.

Kang SC et. al. (August 2015). Transcriptomic Profiling and H3K27me3 Distribution Reveal Both Demethylase-Dependent and Independent Regulation of Developmental Gene Transcription in Cell Differentiation. PLoS One. 10(8):e0135276.

He XB et. al. (December 2014). Vitamin C Facilitates Dopamine Neuron Differentiation in Fetal Midbrain Through Tet1- aND Jmjd3-Dependent Epigenetic Control Manner. Stem Cells.

Suzuki T et. al. (September 2013). Identification of the KDM2/7 histone lysine demethylase subfamily inhibitor and its antiproliferative activity. J Med Chem. 56(18):7222-31.

Wang C et. al. (September 2012). UTX regulates mesoderm differentiation of embryonic stem cells independent of H3K27 demethylase activity. Proc Natl Acad Sci U S A. 109(38):15324-9.

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