Figure 3.

Reconstitution of purified FtsX‐His in nanodiscs. A. SDS‐PAGE of fractions from purification of reconstituted FtsX‐His nanodiscs. Lane 1, FtsX‐His alone; lane 2, MSPE3D1 alone; lane 3, flow through of nanodisc reconstitution from Ni‐NTA column; lane 4, wash 1; lane 5, wash 2; lane 6, wash 3; lane 7, wash 4; lane 8, Ni‐NTA‐column affinity‐purified nanodiscs; lane 9, nanodiscs purified by affinity chromatography followed by gel filtration. Purification of FtsX‐His and nanodisc reconstitution are described in Materials and Methods. B. Elution profile of FtsX‐His nanodiscs in analytical size‐exclusion chromatography (Superdex 200 30/100; buffer = 20 mmol/L Tris‐HCl; 8.0, 100 mmol/L NaCl; flow rate = 0.5 mL per min; A₂₈₀ detection). Nanodiscs eluted at 23.5 min, corresponding to a volume of ≈11.8 mL. Minimal additional peaks were detected elsewhere in chromatograms (data not shown). The experiment was performed independently three times with similar results.