EV-Loc

EV cargo localisation kit for Western blot analysis

Reference K213

EV-Loc kit allows to determine the localisation (i.e. internal or at the surface) of proteins associated with extracellular vesicles (EVs).

Principle

The way to determine the localisation of a protein of interest associated with EVs

EV-Loc kit allows to determine the localisation (i.e. internal or at the surface) of proteins associated with extracellular vesicles (EVs). Thanks to the lipid bilayer, internal proteins are protected from digestion by proteases while surface proteins are not protected. By treating with proteases intact or previously permeabilised EVs, it is possible to determine the localisation of proteins which are known to be associated with 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 EV markers, respectively.

Schematic representation of how EV-Loc kit works.

Specifications

EV-Loc kit is designed for the analysis of EVs of sufficient quality and purity. The purer the EVs, the more reliable the result.

 

The amount of EVs used must be sufficient to detect EV markers and the protein of interest by SDS-PAGE and Western blot.

Strengths of EV-Loc kit

o   Easy to use

o   Reproducible

o   Reliable

Additional information

  K213_2 K213_5
 Size For 2 samples For 5 samples
 Downstream applications Western Blot
 Shelf life 1 month

Data and examples

Pure EGFP EVs (in-EV, Ref. X133) were subjected or not to protease digestion in the presence or not of a permeabilisation agent (Perm.). Blots were probed with antibodies against Alix (EV lumen control), CD81 (EV 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.

EV-Loc

Data Sheet

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