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  • Title: Separation of inner and outer membrane vesicles from Escherichia coli in self-generating Percoll gradients.
    Author: Morein S, Henricson D, Rilfors L.
    Journal: Anal Biochem; 1994 Jan; 216(1):47-51. PubMed ID: 8135365.
    Abstract:
    A rapid and simple method for separating and isolating the inner and outer membranes of Escherichia coli is described. Membrane vesicles were prepared either by passing the bacteria through a French press or by conversion of the cells to spheroplasts by the lysozyme-EDTA treatment and disruption of the spheroplasts by sonication. The membrane vesicles were collected by ultracentrifugation and suspended in a Percoll-containing buffer. The membranes were separated by centrifugation of the membrane-Percoll mixture in a fixed angle rotor at 27,000gmax for 30 min in a preparative centrifuge. One low-density and one high-density band was obtained, corresponding to the inner and outer membranes, respectively. For the membranes prepared by French pressing 69 and 3.3% of the total activity in the gradient of the inner membrane marker NADH-oxidase was found in the low-density and the high-density bands, respectively. For the outer membrane marker 2-keto-3-deoxyoctonate (KDO), 69 and 7.3% of the total amount of KDO in the gradient was found in the high-density and the low-density bands, respectively. For the membranes prepared by sonication of spheroplasts the same figures were 39 and 6.5% for NADH-oxidase and 52 and 9.0% for KDO. The total time of preparation of membrane vesicles, from harvesting the bacteria to the separation of the inner and outer membrane vesicles, is about 6 h. A good separation of the inner and outer membranes was still obtained when samples corresponding to about 10 mg of membrane protein were added to a 33-ml gradient.
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