Bacterial Culture Filtrate Protein Concentration using an Ultrafiltration Device

Abstract

Source: Ryan, J. M., et al. Mycobacterium tuberculosis Extracellular Vesicle Enrichment through Size Exclusion Chromatography. J. Vis. Exp. (183) (2022)

This video demonstrates the isolation and concentration of bacterial culture filtrate proteins, which include bacterial extracellular vesicles, using ultrafiltration. The concentrated extracellular vesicles serve as diagnostic markers for disease detection.

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) centr....

Materials

List of materials used in this article

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