Fluorescent EMSA for DNA-Protein Interactions

Experiments were performed as described in (34 (link),36 (link)). Briefly, double-stranded DNA probes with 5′ Cy5 or FAM labels at one strand were prepared using an annealing buffer (20 mM Tris–HCl, 50 mM MgCl₂, 50 mM KCl, pH 8.0). Purified protein samples and fluorescently labeled DNA were incubated for 1 h at RT in EMSA buffer. Mini gels were first pre-ran in 1× TG buffer (Biorad:1610734) at 200 V for 30 min. Then, samples were loaded, and gels were run for 30 min at 200 V at 4°C. Images are captured using an Amersham Typhoon 5 Biomolecular Imager and quantified using ImageQuantTL 7.0. DNA probes used are listed in (Supplement Table S1). Cooperativity factors for homodimers were calculated as described in (37 (link),38 (link)) and single tube dual color EMSAs with differently labelled DNA elements were performed as described in (39 (link)).