Multi gauge software 3

EMSA was carried out as described previously [65 (link)] with few modifications. Briefly, 500 bp (approximately) DNA from the promoter of flagellar motor protein operon labelled with ATP γ-³²P using Klenow enzyme. Reactions for gel shift assay contained 0.031pM of ³²P -labelled DNA and 500 μM FeCl₃ in the binding buffer (10 mM Tris–acetate, pH 8.0,8 mM MgCl₂, 10 mM potassium acetate, 1 mM DTT, 110 μg poly dI-dC and 3.5% PEG 8000) in final volume of 20 μl. Protein concentrations used were 85 nM, 106 nM, 128 nM, 149 nM, 170 nM, 191 nM, 212 nM, 234 nM, 255 nM and 276 nM for binding to the mot promoter and 112 nM, 263 nM and 360 nM for binding to the xss promoter. For cold competition, unlabelled DNA fragments were added to the reactions. The reactions were incubated at 28°C for 30 min. Reaction were loaded onto a 4% non-denaturing polyacrylamide gel in TBE buffer at 200V for 3h in the cold room and were visualized by Fujifilm’s Multi Gauge software 3.0. Primers used for amplification of probes are listed in Supporting S13 Table.

Purified C. crescentus ParB-His₆ (WT and mutants, at final concentrations of 0.7, 1.5, 3.1, 6.2, and 12.5 µM) were incubated with 5 nM radiolabeled P³²-α-CTP (Perkin Elmer), 30 µM of unlabeled CTP (ThermoFisher), and 1.5 μM of 22 bp parS DNA duplex in the binding buffer (100 mM Tris pH 8.0, 100 mM NaCl, and 10 mM CaCl₂) for 10 min at room temperature. 4 μL of samples were spotted slowly onto a nitrocellulose membrane and air-dried. The nitrocellulose membrane was wrapped in cling film before being exposed to a phosphor screen (GE Healthcare) for 2 min. Each DRaCALA assay was triplicated, and a representative autoradiograph was shown. Data were quantified using Multi-Gauge software 3.0 (Fujifilm), the bound fraction were quantified as described previously (Roelofs et al., 2011 (link)). Error bars represent standard deviations from triplicated experiments.