FtsZ derivatives were purified by precipitation with ammonium sulfate as described (Haeusser et al., 2014 (link)). After resuspension in Storage Buffer (50 mM Tris pH 7.5, 250 mM KCl, 10 mM MgCl2, 1 mM EDTA, and 10% glycerol), GDP (0.05 mM) was added followed by freezing in liquid nitrogen and storage at −80°C.
His6-tagged FtsA and FtsA*-like variants were purified using Talon metal affinity resin as described (Krupka et al., 2017 (link)). The purified proteins (Fig. S6 ) were resuspended in Storage Buffer supplemented with 0.05 mM ADP, frozen in liquid nitrogen and stored at −80°C. The CB-X protein estimation assay (G-Biosciences) was used to determine protein concentration.
Cell extracts were processed for immunoblotting as described (Haeusser et al., 2014 (link)), using anti-FLAG (1:2000), affinity purified polyclonal anti-FtsZ (1:5000), and primary and goat anti-rabbit secondary antibodies (1:2000, Sigma) conjugated to horseradish peroxidase. Western Lightning ECL Pro kit (PerkinElmer) was used to detect chemiluminescence. Protein band intensities were measured and compared using ImageJ (Schneider et al., 2012 (link)).
His6-tagged FtsA and FtsA*-like variants were purified using Talon metal affinity resin as described (Krupka et al., 2017 (link)). The purified proteins (
Cell extracts were processed for immunoblotting as described (Haeusser et al., 2014 (link)), using anti-FLAG (1:2000), affinity purified polyclonal anti-FtsZ (1:5000), and primary and goat anti-rabbit secondary antibodies (1:2000, Sigma) conjugated to horseradish peroxidase. Western Lightning ECL Pro kit (PerkinElmer) was used to detect chemiluminescence. Protein band intensities were measured and compared using ImageJ (Schneider et al., 2012 (link)).
