Isolation of Bacterial Extracellular Vesicles Using Size-Exclusion Chromatography

Take a size exclusion chromatography or SEC column packed with porous resin beads of defined size. It contains a sample reservoir at the top.

Add phosphate-buffered saline or PBS to equilibrate the column beads, and discard the flow-through.

Load a concentrated bacterial culture filtrate that contains extracellular vesicles or EVs, secreted proteins, and contaminants.

EVs are lipid-based nanovesicles that carry bioactive molecules and are secreted by bacteria.

Once the filtrate enters the beads, add PBS to drive it through the resin bed.

Discard the initial eluate containing large non-vesicular contaminants.

Place a collection tube under the outlet and sequentially add PBS.

Large-sized EVs evade the smaller resin pores, move through the interparticle spaces between the beads, and elute first.

Smaller proteins and contaminants enter the pores, follow longer paths, and elute later.

Collect the early-eluting fractions containing EVs and store them for analysis.

For the isolation of the EVs, perform size exclusion chromatography, or SEC, using a small column with a 10-milliliter bed volume for less than 100 milliliters of starting material and a larger column with a 47-milliliter bed volume for