Size Exclusion Chromatography Based Separation of Bacterial Extracellular Vesicles: A Technique to Separate Heterogenous Population of Gram-Negative Bacterial Outer Membrane Vesicles Based on Their Size

Abstract

Source: Collins, S. M., et al., Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity. J. Vis. Exp. (2021).

In this video, we demonstrate a technique–size exclusion chromatography–to separate a heterogenous population of outer membrane vesicles or OMVs derived from bacteria. These vesicles are nanosized spherical structures and are separated into large and small subpopulations based on their size through Size Exclusion Chromatography.

Protocol

1. Preparation of buffers To prepare the ELISA wash buffer, add 3.94 g Tris-base, 8.77 g NaCl, and 1 g bovine serum albumin (BSA) to 1 L of deionized (DI) water. Add 500 µL polysorbate-20. Adjust the pH to 7.2 using HCl or NaOH. To prepare the blocking buffer, add 3.94 g Tris-base, 8.77 g NaCl, and 10 g BSA. Add 500 µL polysorbate-20 to 1 L of DI water. Adjust the pH to 7.2 using HCl or NaOH. To prepare the elution buffer (PBS), add 8.01 g NaCl, 2.7 g KCl, 1.42 g Na2HP…

Materials

Amicon 50 kDa filters	Millipore Sigma	UFC905024
FM 4-64	Thermo Scientific	T13320	1.5 x 50 cm
Glass Econo-Column	BioRad	7371552
Potassium Chloride (KCl)	Amresco (VWR)	0395-500G
Potassium Phosphate Monobasic Anhydrous (KH2PO4)	Amresco (VWR)	0781-500G
Sephacryl S-1000 Superfine	GE Healthcare	17-0476-01
Sodium Chloride (NaCl)	Fisher Chemical	S271-3
Sodium Phosphate Dibasic Anhydrous (Na2HPO4)	Amresco (VWR)	0404-500G
Tris Base	VWR	0497-1KG