A Size Exclusion Chromatography Technique to Purify Mycobacterial Extracellular Vesicles

Abstract

Source: Ryan, J. M., et al. Mycobacterium tuberculosis Extracellular Vesicle Enrichment through Size Exclusion Chromatography. J. Vis. Exp. (2022).

This video demonstrates the size exclusion chromatography (SEC) technique for purifying bacterial extracellular vesicles (EVs) from the culture filtrate of Mycobacterium tuberculosis (Mtb). The technique utilizes a porous resin-packed column to obtain purified fractions of EVs. The porous particles selectively allow smaller proteins to enter the pores, resulting in slower elution. At the same time, EVs are excluded from entering the pores due to their larger size and are eluted first.

Protocol

1. Preparation of crude Mtb EV concentrate

NOTE: For detailed procedures on the cultivation of Mtb and preparation of culture filtrate protein (CFP), see References. It is recommended that bacterial culture media is free from growth supplements with EV-containing or proteinaceous components, such as Oleic Albumin Dextrose Catalase (OADC), and detergents such as Tween. It is also recommended that the bacterial culture's quality and harvested CFP be screened to ensure limited cell death and lysis.

1. Prepare a 100 kDa molecular weight cut-off (MWCO) centrifugal filter (see Table of Materials<....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96-well plate	Corning	15705-066
Automatic Fraction Collector	IZON Science	AFC-V1-USD
Benchtop centrifuge	Beckman Coulter	Allegra 6R
Centricon Plus - 70 Centrifugal filter, 100 kDa cutoff	Millipore Sigma	UFC710008	Ultrafiltration device used in step 1.1
Phosphate-buffered Saline, 1X without calcium and magnesium	Corning	21-040-CV
Pierce BCA Protein Assay Kit	ThermoFisher Scientific	23225
qEV Original 35 nm 5/pk	IZON Science	SP5-USD	SEC column