Bio scale mini bio gel p 6 desalting cartridge

A fully-active MsbA mutant (Supplementary Fig. 11) was expressed and purified as previously described^{6 (link),45 (link)}. Briefly, MsbA expression in BL21 DE3-RILP E. coli cells (Agilent Technologies) was induced for 4 h at 30 °C with 1 mM IPTG at an OD₆₀₀ of ~1. Crude membranes were prepared as described above for HR, and were solubilized for 1 h at RT with 2% DDM and 0.04% sodium cholate in a buffer containing 100 mM NaCl, 20 mM Tris/HCl, with 15% glycerol, 0.5 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and 1 mM PMSF, pH 8. Solubilized MsbA was purified by metal affinity chromatography (Talon Superflow; Clontech) followed by size-exclusion chromatography using a Bio-Scale Mini Bio-Gel P-6 Desalting Cartridge (Bio-Rad, Hercules, CA) equilibrated with storage buffer: 100 mM NaCl, 20 mM Tris/HCl, 0.065% DDM, 0.04% sodium cholate, with 15% glycerol and 0.2 mM TCEP, pH 7.5. MsbA was stored at −80 °C until use. Protein concentration was determined by absorbance at 280 nm and purity was estimated at >95% from SDS-PAGE gels stained with Instant Blue (Expedeon). Reconstitution of purified MsbA into liposomes formed by E. coli polar lipids was performed by gel filtration and extrusion, as described for HR, but using a 1:10 protein:lipid ratio (w/w) in 100 mM NaCl, 20 mM Tris/HCl, with 0.1 mM TCEP, pH 7.4.