Histone Peptide Binding Assay

The dCypher peptide screen assay was performed as previously described [35 (link)]. Briefly, 5 μL of GST-tagged reader domains (optimal protein concentration for library screening determined by initial binding curves to candidate peptides) were incubated with 5 μL of 400 nM (100 nM Final) biotinylated histone peptides (EpiCypher) for 30 min at 23 °C in 1× AlphaLISA Epigenetics buffer + epigenetics buffer supplement (PerkinElmer, AL1008) in a 384-well plate. A 10 μL mix of 5 µg/mL (2.5 μg/mL final) glutathione Acceptor beads (PerkinElmer, AL109M) and 10 μg/mL (5 μg/mL final) streptavidin Donor beads (PerkinElmer, 6760002) was prepared in 1× [Epigenetics buffer + supplement] and added to each well. Plates were incubated at 23 °C in subdued lighting for 60 min and AlphaLISA signal measured on a PerkinElmer 2104 EnVision (680 nm laser excitation, 570 nm emission filter ± 50 nm bandwidth).