Characterization of PaSSB Protein Binding Assay

The EMSA [82 ] was conducted in accordance with a previously described protocol [83 (link),84 (link)]. The EMSA was performed using a LightShift Chemiluminescent EMSA Kit (Thermo Scientific, Waltham, MA, USA) with a minor modification for PaSSB. In brief, PaSSB (0–5 μM) was incubated for 60 m at 37 °C with a DNA substrate (30 fmol/μL) in a total volume of 6 μL in 40 mM Tris-HCl (pH 7.5) and 50 mM NaCl. Following incubation, 4 μL of a dye mixture (0.01% bromophenol blue and 40% glycerol) was added. Native polyacrylamide gel (8%) was pre-electrophoresed at 110 V for 10 min. Thereafter, the resulting samples were loaded and resolved on pre-run gel and electrophoresed at 100 V for 1 h in a TBE running buffer (89 mM Tris borate and 1 mM EDTA). The protein-DNA complexes were electroblotted to a positively charged nylon membrane (GE, USA) at 100 V for 30 min in a fresh and cold TBE buffer. The transferred DNA was cross-linked with a nylon membrane using a UV light cross-linker instrument equipped with 312 nm bulbs for a 10 min exposure. Biotin-labeled DNA was detected using a streptavidin-horseradish peroxidase conjugate and a chemiluminescent substrate contained in a SuperSignal™ West Atto Ultimate Sensitivity Substrate (Pierce Biotechnology, Rockford, IL, USA). The ssDNA binding ability of the protein was estimated through linear interpolation from the concentration of the protein that bound 50% of the input DNA. To assess whether the flavonol inhibited the binding activity of SSB, PaSSB (1.25 μM) with a DNA substrate was individually incubated with myricetin (0, 1.5, 3.1, 6.3, 12.5, 25, 50, 75, and 100 μM), quercetin (12–100 μM), kaempferol (12–100 μM), or galangin (12–100 μM) for 60 m at 37 °C. The resultant protein solution was then analyzed using the EMSA.