Amicon ultra 100k centrifugal filter

Site-directed mutants were constructed using the QuikChange Lightning and Quik Change II XL mutagenesis kits (Agilent) as reported before [29 (link)]. The sequences of the forward primers are listed in Table 1.
Recombinant wild-type and mutant Na⁺-NQR strains were grown in LB (Miller) medium as reported before [29 (link)] in 30-liter cultures in fermenters (New Brunswick BF-5000, Microbiology Core Facility, CBIS, RPI) at 37 °C with constant aeration (20 l per minute) and agitation (300 rpm). The expression of the nqr operon was induced by adding arabinose. Cells were harvested, washed, and broken in buffer (50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM MgCl₂) in the presence of DNase and a cocktail of protease inhibitors. Membranes were obtained by ultracentrifugation (100,000 ×g) and washed with a medium containing 5.0 mM imidazole, 300 mM NaCl, and 0.05% glycerol.
Wild-type and mutant Na⁺-NQRs were purified by Ni⁺-NTA affinity chromatography and anion exchange DEAE chromatography as reported before [16 (link),30 (link)]. For the preparation of LDAO-washed Na⁺-NQR [11 (link),16 (link)], the enzyme stock solution (50 μl) was diluted in a 20-fold volume with a washing buffer (50 mM Tris-HCl, 1.0 mM EDTA, 5% glycerol, and 0.05% LDAO, pH 8.0), concentrated with Amicon Ultra 100 K centrifugal filter (Merck-Millipore, Billerica, MA), diluted 20-fold again, and re-concentrated.