EPIgeneous Binding Domain Discovery kit
A kit to determine how to assess epigenetic reader binding
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 A has been successfully validated on 4 different domains, including some important therapeutic targets, in the BRD family.
Full assay optimization performed on BRD4(1) using EPIgeneous Binding Domain Kit A. Additional data describes the use of EPIgeneous Binding Domain Kit A for the profiling of reference (+)-JQ1 compound on multiple binding domains.
The GST-Reader concentration was fixed at 5nMf while the peptide-biotin was serially diluted (100nMf to 0.005nMf).
For each peptide-biotin concentration, a negative control was performed by removing the GST-Reader protein from the wells. This negative control was used as non specific signal to calculate the HTRF delta ratio. This specific signal was proportional to the specific interaction measured between GST-BRD4(1) and [Lys(5,8,12,16)Ac]-H4(1-21)-biotin peptide.
The 4nM Kd value was determined from this experiment using a one-site specific binding regression (saturation equations).
Peptide-biotin titration performed with various DMSO percentages. The apparent Kd values are determined using one-site specific binding regression (saturation equations). Due to the competitive inhibitor nature of the DMSO on the BRD4(1)/H4 peptide interaction (1), a shift of apparent Kd, is observed while DMSO% increases. As the DMSO competes on BRD4(1)/H4 peptide interaction, the assay window decreases while the DMSO percentage increases. The assay is then recovered by increasing the peptide-biotin concentration. In the case shown here, the optimal peptide-biotin concentration was set between the real Kd and the EC100 obtained for the titration without DMSO, a compromise between assay window and assay sensitivity to enable inhibitor studies (1% DMSO and 3nM peptide-biotin).
The assay was performed using 3 nM peptide-biotin, 5 nM GST-BRD4(1) and 1% DMSO, all set constant throughout the inhibitor titration.
The IC50 of (+)-JQ1 and H4 tetra-acetylated peptide are in good agreement with published data (1, 2).
The assay was performed using 3 nM peptide-biotin, 5 nM GST-BRD4(1) and 1% DMSO.
The 0.76 Z' factor underlines the robustness of the assay and its suitability for HTS.
Robust assay tools to decipher key epigenetic interactions - Flyers
Case study: BRD4 bromodomain - Posters
BMG Labtech - Application Notes
Get the brochure about technology comparison. - Brochures
All-in-one kit for robust detection of Total IRS1