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....

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