Fluorescent Electrophoretic Mobility Shift Assay

Double‐stranded DNA (dsDNA) probes with 5′ Cy5 or Cy3 dyes attached to the forward strand were prepared using an annealing buffer (20 mM Tris/HCl, 50 mM MgCl₂, 50 mM KCl, pH 8.0). Protein samples and fluorescently labeled DNA are incubated for ∼2 h in EMSA buffer (10 mM Tris/HCl pH 8.0, 0.1 mg ml − 1 BSA, 50 µM ZnCl₂, 100 mM KCl, 10% glycerol, 0.10% Igepal CA630, 2 mM βME). Gels were first preran using a 1 × TG buffer (Tris 0.25 mM, glycine 192 mM, pH 8.0) at 200 V for 30 min. Then, 10 µl 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 supplementary table S4, Supplementary Material online. Cooperativity factors were calculated as described in (Ng et al. 2012 (link)) for heterodimers and (Jerabek et al. 2017 (link); Wang et al. 2018 (link)) for homodimers.