Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity

Size exclusion chromatography is a useful approach for separating the subpopulations of heterogeneously-sized bacterial vesicles and for removing nucleic acids and proteins from bacterial supernatants. Size exclusion chromatography is less expensive, faster, and more reproducible than other separation techniques, including density gradient ultracentrifugation. We've demonstrated the potential of this approach using A.actinomycetemcomitans vesicles, but we expect that it could be applied to other heterogeneously-sized bacterial vesicle populations as well.

Be sure to properly degas all of the buffer and bead solutions and to pipette the solution slowly without introducing bubbles but quickly enough that the beads pack homogeneously without drying out. To begin, use a glass rod to mix a stock bottle of gel filtration medium and pour the volume required to fill the column plus approximately 50%excess into a glass bottle. Allow the beads to settle and decant the excess liquid.