Extracellular vesicle separation from milk and infant milk formula using acid precipitation and ultracentrifugation

• Main Authors: Anindya Mukhopadhya, Jessie Santoro, Lorraine O’Driscoll
• Format: Article
• Language: English
• Published: Elsevier 2021-12-01
• Series: STAR Protocols
• Subjects: Health Sciences; Molecular Biology; Protein Biochemistry; Protein expression and purification; Biotechnology and bioengineering
• Online Access: http://www.sciencedirect.com/science/article/pii/S266616672100527X
• ISSN: 2666-1667

Summary: Separation of highly enriched extracellular vesicles (EVs) fractions from milk is desirable for quantification, cargo analysis, functional characterization, and investigation as delivery vehicles for nutrients and/or therapeutics. However, a rigorous, reproducible protocol is lacking. This protocol considers a crucial aspect typically overlooked, i.e., that caseins are of similar size to, but more abundant than, EVs in milk. Our protocol combines acid pre-treatment and gradient ultracentrifugation, producing EV-enriched fractions suitable for downstream orthogonal characterization approaches.For complete details on the use and execution of this protocol, please refer to Mukhopadhya et al. (2021).