Alphalisa epigenetics buffer

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).

All binding assays were conducted in light gray, half area 96-well plates (PerkinElmer, 6002350) in a total volume of 40 μl. All stock solutions were prepared in the assay buffer comprised of 1× AlphaLISA Epigenetics buffer (PerkinElmer, 5× AL008C/F) with 0.05% v/v Tween 20 and 2 μM tris(2-carboxyethyl)phosphine. For initial screens, each recombinant His₆-tagged PBRM1-BD4 mutant was incubated at three concentrations (0.1 μM, 1 μM, and 10 μM) with 50 nM biotinylated histone H3K14ac(1–20) peptide (NH₂-ARTKQTARKSTGGK(acetyl)APRKQLK(biotin)-CONH₂; Peptide 2.0). Ten microliters of a 4 × (200 nM) biotinylated histone H3K14ac peptide stock solution and 10 μl of 4 × (40, 4, 0.4 μM or 0.004–8 μM) protein stock solutions were added to each well. The plate was then incubated for 30 min at room temperature. A bead solution comprising 8 μg/ml streptavidin donor beads and 8 μg/ml nickel-chelate acceptor beads (PerkinElmer, AlphaScreen Histidine (Nickel Chelate) Detection Kit, 500 assay points, 6760619C) was prepared in assay buffer. Twenty microliters of bead solution was added to each well under reduced light, and the plate was covered and incubated for an additional hour in the dark. Luminescence was subsequently read on a BioTek Cytation 5 imaging reader (Agilent, 16277) using the Alpha filter cube (Agilent, 1325000), and Alpha counts were analyzed using GraphPad Prism.