Electrophoretic Mobility Shift Assay Protocol

EMSA was performed according to LightShift Chemiluminescent EMSA Kit Protocols, (Thermo Scientific, United States). In brief, oligonucleotides b-WT (124 bp) from Borrelia burgdorferi strain B31 (Riley et al., 2009 (link)) and cc-WT (200 bp of ybab_Cc gene) from C. crescentus (this work) were labeled at 3′ end with Biotin-11-UTP according to the manufacturer’s protocol (Biotin 3′ end DNA Labeling Kit, Thermo Scientific, United States). Labeled target probes were incubated with increasing concentrations of purified YbaB_Cc in a reaction containing binding buffer (LightShift Chemiluminescent EMSA Kit, Thermo Scientific) and 50% glycerol at 25°C for 30 min. The reactions were electrophoresed in 6% DNA retardation gel (Invitrogen, United States) in 0.5x TBE. Separated DNA products were then electroblotted onto a positively charged Nylon membrane (Thermo Fisher Scientific, United States) using a semi-dry transfer apparatus (Bio-Rad, United States). Cross-linking by UV was done at 120 mJ/cm² for 1 min immediately after electroblotting. Detection of DNA and DNA-protein complexes was carried using Chemiluminescent Nucleic Acid Detection Module (Thermo Scientific, United States).