Bacterial Culture Filtrate Protein Concentration using an Ultrafiltration Device

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.

To begin, prepare a 100 kilodalton molecular weight cutoff centrifugal filter by adding the full volume capacity of PBS. Centrifuge for five minutes at 2,800 times g at 4 degrees Celsius. Discard the flow through, and any remaining PBS, and fill the sample chamber with Mtb CFP to its maximum capacity. Centrifuge at 2,800 times g at 4 degrees Celsius until the volume has reduced to the minimum volume of the ultrafiltration device. Repeat as necessary until the entire sample has been reduced sufficiently.

Add 1x PBS to the filter unit containing the concentrate up to the total device capacity. Reduce the volume as described before and repeat this step five times to ensure complete washing and buffer exchange. Then, recover the 100 kilodalton-concentrated CFP retentate, or 100R, according to the ultrafiltration device specifications. Wash the filter with a minimal volume of 1x PBS at least three times, and pull the wash with the 100R to maximize the recovery.