Bacterial Culture Filtrate Protein Concentration using an Ultrafiltration Device

Protocol

1. Preparation of crude Mycobacterium tuberculosis (Mtb) extracellular vesicle (EV) concentrate

NOTE: For detailed procedures on the cultivation of Mtb and preparation of culture filtrate protein (CFP). 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's quality and harvested CFP be screened to ensure limited cell death and lysis.

1. Prepare a 100 kDa molecular weight cut-off (MWCO) centrifugal filter (see Table of Materials) by adding the full volume capacity of phosphate-buffered saline (1x PBS). Centrifuge for 5 min at 2,800 x g at 4 °C.
   NOTE: If using a filter with no dead stop volume, care must be taken to ensure the sample volume does not reduce below the filter level, resulting in complete filter drying during centrifugation.
2. Discard the flow-through and any remaining PBS before filling the sample chamber with Mtb CFP to its maximum capacity. Centrifuge at 2,800 x g at 4 °C until the volume has reduced to the minimum volume of the ultrafiltration device. Repeat as necessary, adding more CFP to the unit until the entire sample has been reduced sufficiently.
   NOTE: Retain a portion of the 100 kDa flow-through (100F) material for downstream qualification if desired. Store at 4 °C.
3. Add 1x PBS to the filter unit containing the concentrate up to the total device capacity. Reduce the volume as described in 1.2. Repeat this step five times to ensure complete washing and buffer exchange.
4. Recover the 100 kDa concentrated CFP retentate (100R) according to the ultrafiltration device specifications (see Table of Materials). Once the 100R is recovered, wash the filter with a minimal volume of 1x PBS at least three times, and pool the wash with the 100R to maximize the recovery.
5. Quantitate the 100R material with a bicinchoninic assay (BCA) following the manufacturer's instructions (see Table of Materials). To perform the assay, use several dilutions of samples 1:2, 1:5, and 1:10 in PBS. Test the sample in triplicate.
   NOTE: Retain a portion of the 100R material for downstream qualification if desired. Store at 4 °C.

Materials

Name	Company	Catalog Number	Comments
Automatic Fraction Collector	IZON Science	AFC-V1-USD
Centricon Plus - 70 Centrifugal filter, 100 kDa cutoff	Millipore Sigma	UFC710008	Ultrafiltration device used in step 1.1
Phosphate-buffered Saline, 1X without calcium and magnesium	Corning	21-040-CV
Nitrocellulose membrane, Roll, 0.2 μm	BioRad	1620112