CLAMP Protein-DNA Binding Assay

DNA or nucleosome probes at 35 nM (700 fmol/reaction) were incubated with MBP-tagged CLAMP DBD protein or MBP-tagged FL CLAMP protein in a binding buffer. The binding reaction buffer conditions are similar to conditions previously used to test ZLD nucleosome binding (McDaniel et al., 2019 (link)) in 20 µl total volume: 7.5 µl BSA/HEGK buffer (12.5 mM HEPES, pH 7.0, 0.5 mM EDTA, 0.5 mM EGTA, 5% glycerol, 50 mM KCl, 0.05 mg/ml BSA, 0.2 mM PMSF, 1 mM DTT, 0.25 mM ZnCl₂, and 0.006% NP-40) 10 µl probe mix (5 ng poly[d-(IC)], 5 mM MgCl₂, 700 fmol probe), and 2.5 µl protein dilution (0.5µM, 1 µM, and 2.5 µM) at room temperature for 60 min. Reactions were loaded onto 6% DNA retardation gels (Thermo Fisher Scientific) and run in 0.5× Tris–borate–EDTA buffer for 2 hr. Gels were post stained with GelRed Nucleic Acid Stain (Thermo Fisher Scientific) for 30 min and visualized using the ChemiDoc MP imaging system (Bio-Rad).