EPIgeneous Binding Domain kit B HTRF®

This biochemical assay format enables the detection of the interaction between a subset of epigenetic binding domains and modified histone sequences.

High sensitivity
Good DMSO tolerance

Download the protocols

This biochemical assay format enables the detection of the interaction between a subset of epigenetic binding domains and modified histone sequences.

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Overview

The EPIgeneous Binding Domain kit series provides a simple biochemical approach to study epigenetic reader domain interactions with modified histones. All kits are based on a GST-tagged binding domain / biotin-coupled Histone peptide format, and can be run using the same add-and-read single plate protocol.

Binding Domain kit B has been successfully validated on a wide variety of readers (18), including some important therapeutic epigenetic targets.

Benefits

GOOD DMSO TOLERANCE
HIGH SENSITIVITY
LOW PROTEIN OR PEPTIDE CONSUMPTION

Assay principle

In EPIgeneous Binding Domain kit B, the GST-tagged reader domain protein binding to the biotinylated peptide substrate is detected by a conjugate mix: anti-GST Eu cryptate-labeled antibody conjugate (donor), and XL665-conjugated streptavidin (acceptor). The interaction of the reader domain with the substrate brings the donor and acceptor dyes into close proximity, and allows FRET to occur upon light excitation. The specific signal at 665 nm is inhibited when a specific compound prevents the reader domain protein from binding to its substrate.

EPIgeneous Binding Domain kit B principle

Peptide-biotin titration - UHRF1/ histone H3 peptide interaction.

The validation of the EPIgeneous binding Domain B assay was performed using the Tudor Domain UHRF1 binding to [Lys(9)Me3] H3(1-21) peptide. The GST-UHRF1 concentration was fixed at 5 nM while the peptide-biotin was serially diluted.

The HTRF signal was proportional to the specific interaction measured between GST-UHRF1 and peptide-biotin.

The 2nM Kd value was determined from this experiment using a two-site specific binding regression. A slight shift of apparent Kd is observed when DMSO% increases.

EPIgeneous Binding domain assay B binding optimization

DMSO tolerance - peptide-biotin concentration optimization

The assay window slightly decreased as the DMSO percentage increased. The assay window could then be recovered by increasing the peptide-biotin concentration. The optimal peptide-biotin concentration was selected (between real Kd and EC100 obtained on the titration without DMSO) with a compromise between assay window and assay sensitivity for inhibitor studies. Note that the higher the peptide-biotin concentration, the higher the inhibitor IC50. For further study of inhibitors, 1% DMSO and 6nM peptide-biotin conditions were applied.

EPIgeneous Binding domain assay B DMSO tolerance

Inhibitor titration - UHRF1 by reference compound

The EPIgeneous Binding Domain B assay was performed using 4 nM peptide-biotin, 5 nM GST-UHRF1 and 1% DMSO set constant throughout the inhibitor titration.The IC50 of [Lys(9)Me3]-H3(1-21) peptide is in good agreement with published data (Rothbart et al. Nat Struct Mol Biol, 2012 - 2µM)

EPIgeneous Binding domain assay B inhibition by reference compound

Validated binding domains

Three kits (A, B and C) have already been validated and fully optimized on a selection of 28 key binding domains. For non-validated reader domains, a fourth one, the Discovery Kit, enables researchers to profile which of the A, B or C kits is the best assay solution.