Heterogeneity among membrane vesicles of Escherichia coli: effects of production and fractionation techniques.

We have previously shown that Escherichia coli membrane proteins are associated with subpopulations of membrane vesicles beyond the well-defined inner versus outer membrane classification. To separate these vesicles we used a shallow sucrose gradient which differed in many respects from established procedures. Here we compare this revised technique to the classical sucrose density centrifugation procedure. We found that certain manipulations common to the latter obscured heterogeneity among membrane vesicles. The following treatments degraded vesicle separation: growth in rich medium addition of EDTA to buffers, spheroplasting, sonication, pelleting of membranes prior to sucrose gradient centrifugation, overloading the gradient, and long centrifugation times. Some treatments, such as EDTA addition, affected only selected vesicles. When determining protein localization within bacterial membranes experiments should be designed to avoid or at least minimize these manipulations.