EV-Loc, the technique to determine the localisation of proteins associated with Extracellular Vesicles
Because of the lipid bilayer, internal proteins are protected from digestion by proteases while surface proteins are not. It is possible to determine the localisation of proteins which are known to be associated with EVs by treating with proteases intact or previously permeabilised EVs.
Following digestion assay, EV samples are analysed by SDS-PAGE and Western blot. In this kit, antibodies against CD81 and Alix are used to reveal surface and internal EVs markers, respectively.
Specifications
EV-Loc kit is designed for the analysis of EVs of sufficient quality and purity.
Higher purity levels of EVs lead to more reliable results.
To accurately detect EVs markers and the target protein using SDS-PAGE and Western blot techniques, a sufficient quantity of EVs is required.
Shelf-life: At least 3 months at -20°C
Pure EGFP EVs were subjected or not to protease digestion in the presence or not of a permeabilisation agent (Perm.). Blots were probed with antibodies against Alix (EVs lumen control), CD81 (EVs surface control) or pilot peptide, PP (EGFP cargo fused to PP).
Western blot on pure EGFP EVs after protease treatment using EV-Loc kit.
As expected, external CD81 was digested in the presence of the protease, both on intact or permeabilised EVs, while internal Alix was digested only when EVs were permeabilised. For the protein of interest, here EGFP, the same pattern as Alix was obtained, revealing the internal localisation of EGFP protein.