Size Exclusion Chromatography Based Separation of Bacterial Extracellular Vesicles: A Technique to Separate Heterogenous Population of Gram-Negative Bacterial Outer Membrane Vesicles Based on Their Size

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 1 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 2 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 2 milliliters of a 100 to 200-nanomoles 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 1-milliliter fractions in each tube. While collecting the samples, continuously add elution buffer to the column. To clean the column, run 1 column volume of 0.1 molar sodium hydroxide through the column, followed by 2 column volumes of elution buffer.