Fig. 1

Characterization of the H3K23Ac peptide ligand for the biochemical TRIM24 AlphaScreen assay development. a Binding affinity (K _D ) of the His-TRIM24-PB/H3K23Ac-peptide interaction calculated from kinetic fitting of the association and dissociation profiles measured by biolayer interferometry. b AlphaScreen signal to background (S/B) for His-TRIM24-PB binding to the biotinylated-H3K23Ac peptide (1 µM) using the indicated amounts of wild-type (WT) and bromodomain mutant (N980A) protein. c AlphaScreen assay optimization using a matrix of TRIM24 protein and peptide (i.e., twofold dilution series starting from 20 to 60 nM, respectively). The Alpha Signal (S/B) is the average of 2 independent 384-well plates highlighting conditions (red box) selected for the final assay protocol (Additional file 1: Table S1). d AlphaScreen peptide competition and dose–response validation. Non-biotinylated H3K23Ac peptide was titrated into a solution of His-TRIM24-PB (5 nM) and biotinylated-H3K23Ac peptide (15 nM) with IC₅₀ curves shown from 6 independent plates