Superdex 200 pg column

RBD-His protein was expressed in Expi293 cells and purified on Ni-NTA resin (#88221, Thermo Fisher) followed by size-exclusion chromatography on a Superdex 200 gel filtration column in PBS (Amanat et al., 2020 (link)). S1-S2-His (referred as S) protein from Wuhan-Hu-1 strain and RBD from variants (Alpha, Beta, Gamma, and Delta) were expressed baculovirus-free in High Five insect cells (Bleckmann et al., 2019 (link)) and purified on HisTrap excel column (Cytiva) followed by preparative size exclusion chromatography on 16/600 Superdex 200 pg column (Cytiva) (Bertoglio et al., 2021 (link); Korn et al., 2020 (link)).

Purifications of MinD, MinD D40A and MinE were performed as previously described⁵² and all used plasmid constructs are indicated in Supplementary Table 4. Divalent streptavidin was assembled as outlined in Howarth et al.⁵³ , both pET21a-Streptavidin-Alive (Addgene plasmid no. 20860) and pET21a-Streptavidin-Dead (Addgene plasmid no. 20859) were a gift from A. Ting^{35 (link)}. Biotin-modification of aldolase (no. 28403842, Cytiva) with EZ-Link Maleimide-PEG2-Biotin (no. A39261, Thermo Fisher Scientific) was achieved through incubation at room temperature for 1 h and subsequent size-exclusion chromatography on a 16/600 Superdex 200 pg column (Cytiva), equilibrated in storage buffer (50 mM HEPES pH 7.25, 150 mM KCl, 0.1 mM EDTA, 10% glycerol, 0.4 mM TCEP), using an Äkta Pure chromatography system (Cytiva). LC–MS and SDS–PAGE was performed to assess purity and integrity of all purified or modified proteins. A customized Bradford assay (no. 5000006, Bio-Rad Protein Assay; Bio-Rad Laboratories Inc.) was used to determine protein concentrations and single-use aliquots were flash frozen in liquid nitrogen and stored at −80 °C.