Isolation of Bacterial Extracellular Vesicles Using Size-Exclusion Chromatography

JoVE Encyclopedia of Experiments

Microbiology

0 weergaven • 3:24 min • September 26th, 2025

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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 more than 100 milliliters of starting material. Over several hours before the experiment, bring the SEC column and PBS to room temperature, followed by stabilizing the column in a vertical position using a standard laboratory stand and holder. Before connecting to the SEC column, hydrate the sample reservoir by allowing five milliliters of PBS to flow through the frit into a waste container.

Then, unscrew the inlet cap of the column to add two milliliters of PBS to the sample reservoir and carefully connect the reservoir to the column as the PBS is dripping out through the frit. For equilibration of the column, add 47 milliliters of PBS to the sample reservoir, followed by uncapping the bottom of the SEC column. Then, allow all the loaded sample buffer to flow through the column and discard the flow-through.

After loading two milliliters of the sample onto the sample reservoir, allow the sample to enter the column completely and collect the flow-through. Immediately add PBS to the sample reservoir at a volume of 14.25 milliliters minus the sample volume to allow the solution to flow through the column, while discarding the amount equal to the column void volume. Next, position a two-milliliter low-binding microtube directly below the SEC column to collect the first flow-through or fraction one after allowing two milliliters of PBS to run through the column.

Continue to add two milliliters of PBS to the sample reservoir to collect each subsequent fraction. Store the collected fractions at four degrees Celsius for short term storage or minus 80 degrees Celsius for long term storage.

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Laatst bijgewerkt: 22 augustus 2026