Electrophoretic mobility shift assay

An amount of 4–8 nM 5′-³²P-labeled RNAs (indicated by asterisk [*]) were mixed optionally with an equal amount of repZ and incubated for 5 min at 37°C in reaction buffer (25 mM Tris/HCl pH 7.4, 150 mM NaCl, 1 mM MgCl₂). An amount of 0.1 mg/mL yeast RNA (10 mg/mL, Merck) was added. Final concentration of 0-5 µM FopA was added and incubated for 15 min at 37°C. Subsequently, 3 µL 5× native loading dye (0.5× TBE, 50% [v/v] glycerol, 0.2% [w/v] xylene cyanol, 0.2% [w/v] bromphenol blue) was added and loaded immediately on a running native 6% polyacrylamide/0.5× TBE gel and run for 2–3 h at 40 mA at 4°C. The gel was dried on filter-paper. The membrane was wrapped in foil and exposed to a photostimulatable phosphor plate and the signal was detected by the Typhoon 7000 phosphoimager (GE Healthcare).