HTRF EPIgeneous Binding Domain B Detection Kit, 500 Assay Points

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.

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.

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.

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)