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.

Resuspend the beads in elution buffer to a final solution of approximately 70%gel and 30%buffer and degas the solution under vacuum. Mount the column on a ring stand in the vertical position and fill the column with elution buffer to wet the walls before draining the buffer until only about one centimeter remains. Then carefully pipette the gel beads into the column while simultaneously draining the excess buffer to prevent the beads from settling until the column is packed to a height of approximately two centimeters below the bottom of the column reservoir.

Before loading the sample, degas the elution buffer under vacuum. Wash the column with approximately two times the column volume of elution buffer. Once the buffer reaches the top of gel layer, carefully add two milliliters of a 100 to 200 nanomolar per liter outer membrane vesicle sample on top of the gel layer without disturbing the surface and let the sample enter the gel.

Slowly add the elution buffer to the column without disturbing the gel layer and place a 50 milliliter tube under the column. To prevent the column from drying, simultaneously add elution buffer to the top of the column. After collecting 20 milliliters of eluate, place a rack of 1.5 milliliter tubes under the column.

Collect a total of 96 one-milliliter fractions in each tube. While collecting the samples, continuously add elution buffer to the column. To clean the column, run one column volume of 0.1 molar sodium hydroxide through the column followed by two column volumes of elution buffer.

To measure the lipid concentrations, pipette 50 microliters of each fraction into a 96 well plate. Add 2.5 microliters of lipophilic dye to each well. After 15 seconds, measure the florescence intensity.

To measure the concentration of a protein of interest, add 100 microliters of each fraction into individual wells of an ELISA immuno plate. After three hours at 25 degrees Celsius, decant the plate contents. Wash the plate five times with 200 microliters of ELISA wash buffer per wash decanting the plate after each wash.

Add 200 microliters of blocking buffer to each well for a one hour incubation at 25 degrees Celsius. At the end of incubation, decant the plate and add 100 microliters of primary antibody in blocking buffer for an overnight incubation at four degrees Celsius. The next day, decant the plate and wash the plate five times with ELISA wash buffer as demonstrated.

After the last wash, add 100 microliters of secondary antibody in ELISA wash buffer to each well for a one hour incubation at 25 degrees Celsius. At the end of the incubation, wash the plate as demonstrated and add 100 microliters of TMB solution to each well for a 15 to 30 minute incubation at room temperature. When a blue color develops, add 50 microliters of stopping solution to each well and measure the absorbance at 450 nanometers.

After size exclusion column purification of an A.actinomycetemcomitans culture supernatant as demonstrated, two distinct lipid peaks were observed, corresponding to the large and small outer membrane vesicles produced by this strain. ELISA analysis of the sample fractions revealed that the toxin is associated primarily with the subpopulation of large outer membrane vesicles. Immuno blot analysis gave similar but more noisy results, indicating that this approach is less sensitive than ELISA.

As observed, this chromatography technique is able to remove significant amounts of free proteins from the outer membrane vesicles'preparations. However, it is important to note that the total protein concentration does not necessarily correlate with the concentration of specific proteins. It is important to be very careful during the packing and running of the column to avoid bubbles and to prevent the column from drying out.

Dynamic light scattering, particle tracking, and bicroscopy can be performed following size exclusion chromatography to determine the size and other characteristics of the separated outer membrane vesicles.