High-throughput homogeneous epigenetics assays using HTRF technology and the SpectraMax Paradigm microplate detection platform

Olsen C, Pierre N, Gonzalez-Moya A, Graham S, Degorce F


Molecular Devices, Sunnyvale, CA . Cisbio US, Bedford, MA, USA.

5th HTRF Symposium, Avignon, France/ SLAS 2013, Orlando, Florida, USA.

Histone proteins are dynamic proteins that form the scaffold for DNA packaging and also undergo a variety of post-translational modifications that help to determine which areas of the genome are active or inactive. Histone methylation is a post-translational modification that occurs on arginine and lysine residues and that represents one of the most studied post-translational modifications involved in epigenetic regulation. On histone H3, several lysines are potential targets for methyltransferases and demethylases, enzymes that convert these residues to mono-, di-, or tri-methylated marks. Over the past decade, these modifications have been shown to have a prominent role in a broad variety of diseases, and the related enzymes and chromatin interacting proteins are now considered as important druggable therapeutic targets in the drug discovery field. Cisbio Bioassays has generated a panel of reagent tools using the HTRF® technology based on time-resolved fluorescence energy transfer (TR-FRET) between europium cryptate (donor) and XL665 (acceptor). Cisbio Bioassays epigenetic reagent toolbox consist of specific anti-methylated histone H3 antibodies labeled with europium cryptate and streptavidin-XL665 (SA-XL665) that can be combined to form a pair of reagents able to detect a specific methylation state on a histone substrate biotinylated. Each anti-histone HTRF® antibody is specific for a single histone H3 lysine residue and methylation mark enabling development of assays with the highest levels of specificity and sensitivity. This poster features data showing the optimization of G9a (histone methyltransferase) and LSD1 (histone demethylase) assays on the SpectraMax® Paradigm® microplate detection platform. Enzyme titration and time course, cofactor and substrate Km, and IC50 for known inhibitors are demonstrated. These results indicate that HTRF® G9a methyltransferase and LSD1 demethylase assays are suitable for high throughput screening of inhibitors of these disease-relevant enzymes.

HTRF® Technology, HTRF microplate readers

Epigenetic toolbox reagents