Electrophoretic Mobility Shift Assay for MalR

As part of the investigation of the binding properties of MalR and as an in vitro verification of the results obtained by ChAP-seq analysis, EMSAs were performed. Therefore, 100 bp DNA fragments, centering the peak maximum of each particular promoter region, were amplified using PCR (oligonucleotide sequences are given in the Supplementary Table S3C) and analyzed and purified using an agarose gel with subsequent gel extraction with the “PCR clean-up and Gel extraction” Kit from Macherey-Nagel (Düren, Germany). A total of 90 ng of DNA per lane was incubated with different molar excesses of purified MalR protein (threefold and 10-fold molar excess) for 30 min in bandshift-buffer [50 mM Tris–HCl, 5 mM MgCl₂, 40 mM KCl, 5% (v/v) glycerol, pH 7.5]. Subsequently, samples were separated using a 10% native polyacrylamide gel electrophoresis as described previously (Pfeifer et al., 2016 (link)).