Encyclopedia of Experiments: Immunology
A subscription to JoVE is required to view this content.
Transcript
Take a large-volume centrifugal filter device containing vertical regenerated cellulose membranes of a suitable pore size.
Add buffer and centrifuge. Remove the flow-through containing membrane humectant.
Load the device with bacterial culture filtrate protein, or CFP, encompassing soluble proteins and extracellular vesicles, or EVs – nanosized lipid-bound structures containing biomolecules secreted by bacteria.
During centrifugation, CFP passes tangentially across the membranes via centrifugal force.
Biomolecules smaller than the membrane's pores pass through with culture fluid, while larger ones, such as EVs, pass over the membrane surface, minimizing membrane clogging.
The filter concentrates the CFP into a minimum device volume, aided by its dead-stop volume to prevent biomolecule drying.
Add buffer and centrifuge.
The buffer replaces residual media components, creating an isotonic environment for CFP biomolecules and maintaining their integrity.
Place the concentrate cup over the filter cup, invert, and centrifuge. Recover the concentrated CFP retentate for further analysis.
