M2 affinity agarose gel

S. cerevisiae strain BJ2168 was grown in 11 liter yeast extract peptone dextrose medium (YPD; BioShop) in a Microferm fermenter (New Brunswick Scientific) at 30°C, with aeration of 34 cubic feet per hour, and stirring at 300 rpm. Yeast were harvested after 18 h (OD₆₆₀ = 4.5) by centrifugation at 4,000 × g for 15 min at 4°C. All subsequent steps were performed at 4°C. Cell walls were broken by bead beating in lysis buffer (phosphate-buffered saline, pH 7.4, 8% [wt/vol] sucrose, 2% [wt/vol] sorbitol, 2% [wt/vol] glucose, 5 mM ɛ-amino-n-caproic acid, 5 mM benzamidine, 5 mM EDTA, and 0.001% [wt/vol] PMSF). Cellular debris was removed by centrifugation at 3,000 × g for 10 min, and cell membranes were collected by ultracentrifugation at 152,957 × g for 40 min. The membrane pellet was resuspended in 36 ml lysis buffer, divided into four aliquots, flash-frozen in liquid N₂, and stored at −80°C. Two membrane pellets (corresponding to half a fermenter growth) were thawed and solubilized with addition of n-dodecyl-β-D-maltopyranoside (Anatrace) to 1% (wt/vol) final concentration, and n-dodecyl-β-D-maltopyranoside–solubilized S. cerevisiae V-ATPase was isolated with M2 Affinity agarose gel (Sigma-Aldrich) preloaded with SidK_1-278-3×FLAG as described previously (Abbas et al., 2020 (link)). Protein purity was confirmed by SDS-PAGE using 4–20% Mini-PROTEAN TGX protein Gels (Bio-Rad).