Isolation of Bacterial Extracellular Vesicles Using Size-Exclusion Chromatography

Abstract

Source: Watson, D. C., et al. Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria. J. Vis. Exp. (2021)

This video demonstrates the use of size exclusion chromatography with porous resin beads to isolate bacterial extracellular vesicles (EVs) from a bacterial culture filtrate. Larger EVs elute early by bypassing the resin pores, while smaller proteins enter the pores and elute later.

Protocol

1. Size exclusion chromatography (SEC)
NOTE: SEC is used to increase the purity of extracellular vesicles (EVs) and remove non-vesicular protein.

1. Use a small SEC column (10 mL bed volume) for the isolation of EVs from <100 mL of starting material and a larger column (47 mL bed volume) for the isolation of EVs from >100 mL of starting material.
   NOTE: The example below will list volumes for the larger column, with volumes for the smaller column in parentheses.
2. Bring the SEC column and phosphate-buffered saline (PBS) to room temperature over several hours. Stabilize the SEC column in a vertical position using a standard laboratory stand and holder. Alternatively, use commercial chromatography column stands.
3. Before connecting to the SEC column, hydrate the sample reservoir by allowing 5 mL of PBS to flow through the frit and into a waste container. Unscrew the inlet cap of the SEC column, add 2 mL of PBS to the sample reservoir, and carefully connect the reservoir to the column as the PBS is dripping out through the frit (not applicable for small SEC columns). NOTE: This previous step prevents any air bubbles from getting trapped at the top of the SEC column. If air is trapped, remove the reservoir, tap the column to get the air bubble out, and repeat the connection procedure. For the smaller column, simply uncap the top of the SEC column, and attach the sample hopper.
4. Add 47 mL (10 mL) of PBS to the sample reservoir and uncap the bottom of the SEC column. Allow all the loaded sample buffer to flow through the column for equilibration. Discard the flow-through.
5. Load a maximum of 2 mL (0.5 mL) of sample onto the sample reservoir, discard the flow-through, and allow the sample to enter the column completely.
6. Immediately add PBS to the sample reservoir or hopper at a volume of 14.25 mL minus the sample volume (3 mL minus the sample volume, for the small column). Allow the solution to flow through the column and discard this amount equal to the column void volume.
   NOTE: For a typical 2 mL sample, the amount of PBS to be added to the sample reservoir or hopper will be 12.25 mL.
7. Position a 2 mL low-binding microtube directly below the SEC column. Immediately add 2 mL (0.5 mL) of PBS to the sample reservoir and allow it to enter the column. Label the first 2 mL (0.5 mL) of flow-through as Fraction 1. Continue to add 2 mL (0.5 mL) at a time to the sample reservoir to collect each subsequent fraction.
   NOTE: Most bacterial EVs elute in the first 5 fractions. During optimization, the first 12 fractions were collected.
8. Store the fractions at 4 °C for short-term storage (days) or -80 °C for long-term storage.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
qEV original, 35 nm	Izon		Maximal loading volume of 0.5 mL
qEV rack	Izon		For use with the qEV-original SEC columns
qEV-2, 35 nm	Izon		Maximal loading volume of 2 mL
Protein LoBind, 2.0 mL, PCR clean tubes	Eppendorf	30108450