Superdex 75 pc3.2 column

50 μl samples containing full-length CI protein at 125 μM, RexA protein at 125 μM, or a 1:1 mixture of both proteins was incubated for 15 minutes at room temperature and analyzed by SEC using a Superdex 200 PC 3.2 column (GE Healthcare) equilibrated in SEC buffer (20 mM HEPES, pH7.5, 150 mM KCl, 5 mM MgCl₂, and 1mM DTT). To assess CI and RexA interactions with DNA, samples containing the annealed double-stranded DNA substrates OR₁-OR₂ or OL₁-OL₂ were also prepared at 2:1.2 protein to DNA molar ratio. Individual DNA substrates were injected alone at a concentration of 75 μM for comparison. All eluted fractions were further analyzed by SDS-PAGE using 4 –20% gradient gels, and then silver-stained to visualize DNA and Coomassie-stained to visualize protein. Samples were similarly prepared for the D197G, NTD, and CTD CI constructs but were analyzed using a Superdex 75 PC 3.2 column (GE Healthcare) equilibrated in SEC buffer.

OTP86^DYW was analysed by analytical size-exclusion chromatography on a Superdex 75 PC3.2 column (GE Healthcare, Unicorn Software 5.20) in size-exclusion buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl) at a flow rate of 50–70 μl min^–1. Eluted fractions were analysed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) or subjected to DSF. Calibration chromatograms for the column were obtained from GE healthcare online support.