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[Background] Studying of membrane enzymes often requires embedding them into the natural lipid environment. Inside-out, everted membrane vesicles allowed to explore effects of different substrates, inhibitors and other ligands on the operation of these enzymes. Functioning proton pumps, such as NADH: ubiquinone oxidoreductase Type 1 (Complex I), quinol oxidase, and ATPase also requires good electrical sealing of the vesicles. This protocol provides sufficient results in preparation of such vesicles for studying these enzymes (e.g., Euro et al., 2008; Belevich et al., 2011).

Preparation of Everted Membrane Vesicles from Escherichia coli Cells