DNA-Protein Binding Assay with PhoP

Sequences of the DNA probes are listed in Table S3. DNA fragments (200 ng) were incubated with 0, 2, 4 or 8 μM purified His-tagged PhoP in a 20-μL binding reaction system [50 mM Tris, pH 7.9, 50 mM NaCl, 0.5 mM EDTA, 10% glycerol, 1% (v/v) NP-40 (Solarbio, Beijing, China)] at room temperature for 30 min [37 (link)]. The binding mixtures were loaded onto an 8% native polyacrylamide gel in 1×Tris-borate-EDTA (TBE) buffer (0.044 M Tris, 0.044 M boric acid, 0.001 M EDTA, pH 8.0) that had been pre-run on ice at 100 V for 1 h. The electrophoresis was performed on ice at 100 V for 75 min. The gel was stained with ethidium bromide in 1×TBE buffer for 5–10 min. The bands were visualized with a molecular imager ChemiDoc TM XRS+ (Bio-Rad, Hercules, CA, USA).