EPIgeneous Binding Domain kit C HTRF®
- High sensitivity
- Good DMSO tolerance
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.
- GOOD DMSO TOLERANCE
- HIGH SENSITIVITY
- LOW PROTEIN OR PEPTIDE CONSUMPTION
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.
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.
Robust assay tools to decipher key epigenetic interactions - Flyers
Case study: BRD4 bromodomain - Posters
BMG Labtech - Application Notes
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