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.

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.

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.

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.