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

STAR Protoc. 2021 Sep 15;2(4):100821. doi: 10.1016/j.xpro.2021.100821. eCollection 2021 Dec 17.

Abstract

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).

Keywords: Biotechnology and bioengineering; Health Sciences; Molecular Biology; Protein Biochemistry; Protein expression and purification.