Encyclopedia of Experiments: Immunology
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Transcript
Take a size exclusion chromatography column packed with an inert matrix of polysaccharide resin particles. The porous particles have a defined pore size.
Flush the column with an equilibration buffer for optimal purification.
Add a concentrated culture filtrate processed from a culture of Mycobacterium tuberculosis — a pathogenic bacteria. The filtrate contains extracellular vesicles, or EVs, and other soluble proteins secreted by the bacteria.
EVs are heterogeneously sized, small, spherical structures. They contain macromolecules and virulence factors enclosed in a lipid bilayer.
The sample enters the matrix under gravity. Add the buffer to allow sample movement through the column.
The EVs cannot enter the resin due to the small pore size and instead move through the interparticle space, traveling a shorter path and eluting first.
Proteins, having a smaller size, enter the pores, travel a longer path through the resin, and elute at the end.
Collect the fractions containing EVs for downstream analysis.
