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

Under suitable culture conditions, gram-negative bacteria release cargo-carrying outer membrane vesicles, or OMVs, into the supernatant. These vesicles are heterogeneously sized, small, spherical structures, which mediate cellular communication.

To fractionate differently-sized sub-populations of OMVs, take an empty size-exclusion chromatography column with an appropriate filter at its base to support the matrix.

Pack the column with gel filtration matrix consisting of chemically inert, spherical beads of defined pore size, ensuring the size is optimal to exclude large-sized OMVs from entering. Equilibrate the column with an appropriate buffer to prevent the beads from drying for optimum purification.

Load the bacterial culture supernatant containing differently-sized subpopulations of OMVs, protein aggregates, and nucleic acids on the top of the column. Allow the samples to run by the force of gravity.  

As the samples pass through the column, larger OMVs which cannot enter pores, move through the interparticle spaces between the gel beads to get eluted first. Meanwhile, the smaller OMVs penetrate the matrix pores, take a longer path, and elute later.

Due to their smaller size, the protein aggregates, and nucleic acids enter various pores in the matrix, causing them to have the longest retention time in the column and elute as the last fractions. Collect separate OMV eluates and store them for further analysis.