PsrA-DNA Binding Assay Protocol

PsrA-DNA binding reactions (8 μl) contained 30 mM Tris-HCl, pH 7.6, 1 mM DTT, 0.25 mM EDTA, 125 mM NaCl, 0.5 mg/ml BSA, 10 mM MgCl₂, 1 mM CaCl₂, 1.25% glycerol, 4 nM radiolabeled IR1.2 DNA or 6 nM radiolabeled IR1.1 DNA, and different concentrations of PsrA. Reactions were incubated at room temperature for 20 min. Then, protein-DNA complexes were treated with DNase I (0.01 units) for 5 min at the same temperature. DNase I digestion was stopped by adding 1 μl of 250 mM EDTA. Four microlitres of loading buffer (80% formamide, 1 mM EDTA, 10 mM NaOH, 0.1% bromophenol blue, and 0.1% xylene cyanol) was added to the reaction mixtures. Samples were heated at 95°C for 5 min, and immediately chilled on ice before loading onto 8 M urea-6% polyacrylamide gels. After running, gels were dried and exposed to a Phosphorimager screen (Fujifilm, Japan). The radioactive intensity was visualized by a Fujifilm Image Analyser FLA-3000 and was quantified using the Quantity One software (Bio-Rad) (Solano-Collado et al., 2013 (link); Ruiz-Cruz et al., 2018 (link)).