2.8. Biolayer Interferometry (BLI)
In order to further assess binding to SPOTed peptides we employed biolayer interferometry against a commercially available set of biotinylated histone peptides (AltaBioSciences, York, UK Histone array, Set 4 Histone Acetyl-Lysine library), covering the same modifications studied in our membrane SPOTs, using the Octet RED384 system (FortéBio, Portsmouth, UK). Experiments were performed at 25 °C in BLI buffer (20 mM HEPES, pH 7.5, 150 mM NaCl and 0.5 mM TCEP) using the FortéBio data acquisition software V.7.1.0.100. Biotinylated peptides were first immobilized onto Super Streptavidin biosensors (SuperStreptavidin (SSA) Dip and Read Biosensors for kinetic #18-0011, FortéBio, Portsmouth, UK), pre-equilibrated in the BLI buffer then quenched in a solution of 5 µM Biotin (baseline equilibration 60 s, peptide loading for 240 s, quenching for 60 s, 1000× rpm shake speed, at 25 °C). The immobilized peptides were subsequently used in association and dissociation measurements performed within a time window of 600 s (base line equilibration 60 s, association for 600 s, dissociation for 600 s, 1000× rpm shake speed, at 25 °C). Interference patterns from peptide-coated biosensors without protein were used as controls. After referencing corrections, the subtracted binding interference data were analyzed using the FortéBio analysis software (FortéBio data analysis software V.7.1.0.38) provided with the instrument following the manufacturer’s protocols.