EPIgeneous Binding Domain kit C 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 C has been successfully validated on 4 different domains, including some important therapeutic targets, in the BRD and ATAD families.

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 Tb 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 C principle

Peptide-biotin titration - ATAD2 / histone H4 peptide interaction.

The validation of the EPIgeneous binding Domain C assay was performed using Bromodomain ATAD2A assay which measures the interaction of ATAD2A with [Lys(5,8,12,16)Ac] H4(1-21) peptide. The GST-ATAD2A concentration was fixed at 5 nM while the peptide-biotin was serially diluted.

The HTRF signal is proportional to the specific interaction between the two partners.

The 400 nM Kd value was determined using a two-site specific binding regression. A slight shift of apparent Kd is observed while DMSO% increases, likely due to the competitive inhibitor nature of DMSO on this interaction.

EPIgeneous Binding domain assay C binding optimization

DMSO tolerance - peptide-biotin concentration optimization

Due to the competitive nature of DMSO, the assay window decreased as the DMSO % increased. The assay window can then be recovered by increasing the biotinylated peptide concentration. The best compromise was found between the real Kd and EC100 obtained in DMSO free conditions.

Note that the the higher the biotin-peptide concentration, the higher the inhibitor IC50. 1% DMSO and 300 nM were considered for further experiments.

EPIgeneous Binding domain assay C DMSO tolerance

Inhibitor titration - ATAD2A by reference compound

The HTRF assay was performed using 100 nM peptide-biotin, 5 nM GST-ATAD2A and 1% DMSO set constant throughout the inhibitor titration. As expected, the [Lys(5,8,12,16)Ac] H4(1-25) peptide displayed low affinity for ATAD2A.

EPIgeneous Binding domain assay C 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.