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Epigenase JMJD2 Demethylase Activity/Inhibition Assay Kit (Fluorometric)

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Suggested Workflow
Nuclear Protein Extraction
 
 
Histone Demethylase Assay
 
Demonstration of high sensitivity of the JMJD2 activity assay achieved by using recombinant JMJD2 with the Epigenase™ JMJD2 Demethylase Activity/Inhibition Assay Kit (Fluorometric).
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-3081-4848 assays $338.00 
P-3081-9696 assays $549.00 
Availability: Usually Ships In 1-2 Days 
Product Overview

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.

  Epigenase Kit Other Supplier's Kit
Assay Principle Immunoaffinity-based end product measurement Formaldehyde release-based by-product detection
Assay Format 96-well microplate 96-well microplate
Sensitivity High; 5 ng (1 nM) of enzyme detection limit Very low; 10 µg (2 µM) of enzyme detection limit
S/N Ratio >20 with very low background <4 with high background
Interference by Chemical Reagents None Affected by various detergents, solvents, and irons
Use of Cell Lysates Yes No
Specific Enzyme Activity Quantification Yes No
In Vivo Enzyme Inhibition Assay Yes No
Amount of Substrate Required for Activity/Inhibition Assay

<0.5 µM per assay point

50-200 µM per assay point
Amount of Purified Enzymes Required for Inhibitor Screening 20-50 ng per assay point 10-20 µg per assay point
Accuracy of Enzyme Inhibition Assay High (direct detection of changes in amount of end product) Low (indirect detection of formaldehyde release)

Table 1. Comparison of the Epigenase JMJD2 Demethylase Activity/Inhibition Assay Kit and a competing supplier's kit. Competitor data obtained from supplier's website or manual.

 
Fig. 1. Schematic procedure for the Epigenase™ JMJD2 Demethylase Activity/Inhibition Assay Kit (Fluorometric).

Fig. 2. Demonstration of high sensitivity of the JMJD2 activity assay achieved by using recombinant JMJD2 with the Epigenase™ JMJD2 Demethylase Activity/Inhibition Assay Kit (Fluorometric).

Product Components

JB1 (10X Wash Buffer)
JB2 (JMJD2 Assay Buffer)
JB3 (JMJD2 Substrate, 50 µg/ml)*
JB4 (JMJD2 Assay Standard, 50 µg/ml)*
JB5 (Capture Antibody, 1000 µg/ml*)
JB6 (Detection Antibody, 400 µg/ml)*
JB7 (Fluoro-Developer)*
JB8 (Fluoro-Enhancer)*
JB9 (Fluoro-Dilution)
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.

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