eoe sub articles

EoE Sub Articles

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&#39;s quality and harvested CFP be screened t...

a size exclusion chromatography technique to purify mycobacterial extracellular vesicles

Source: Ryan, J. M., et al. <a href="https://app.jove.com/v/63895">Mycobacterium tuberculosis Extracellular Vesicle Enrichment through Size Exclusion Chromatography.</a> 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.

A Size Exclusion Chromatography Technique to Purify Mycobacterial Extracellular Vesicles

1. Preparation of crude Mtb EV concentrate
NOTE: For detailed procedures on the cultivation of Mtb and preparation of culture filtrate protein (CFP), see ...

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 &#160;&#8212; 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.&#160;