Hi, 

In terms of detection of the commonly used CD9, CD63 and CD81 tetraspanin proteins we have detection Abs which are working very well - they have been through extensive testing. 

• ·         Exosome – anti-Human CD9 (for Western), Cat No. 10626D
• ·         Exosome – anti-Human CD63 (for Western), Cat No. 10628D
• ·         Exosome – anti-Human CD81 (for Western),Cat No. 10630D

feel free to contact me at [email protected] for more information

kind regards

ketil

Hi, I agree with Ketil, but you can also check for TSG101 and Alix, and this protein have a function as MVB formation and Rab5b as membrane transport and fusion.

Totally agree with both Ketil and Arief. I suggest you to take a look a this ISEV's article.

kind regards,

FM

thats a great article, agree! sums up what you need to include when characterizing your exosome preparation before digging into the detailed analysis. In summary, it is recommended to include targets which are anchored in the membrane such as the tetraspanins such as CD9, CD63, CD81 or cell adhesion molecules integrins, heterotrimeric G proteins but also proteins with membrane binding capacity (Tsg101, annexins, rabs, scaffolding proteins). this should verify the presence of membranes in the preparation.  Equally important is to label for targets not expected to be there to document the level of contaminating vesicles (golgi: GM130, ER: Hsp90B1, calnexin, histons etc). the presence of membranes and membrane proteins should further be confirmed by EM and also in combination with labeling. this will also act as a support for the measurement of the average size and concentration of exosomes measured e.g. by nano particle tracking analysis, The use of flow cyt can also be used as part of the early exosome verification process prior to detailed analysis by pulling out exosomes from your preparation using e.g. Dynabeads CD9 or Dynabeads CD81 followed by staining and analysis. can also be done directly in the cell culture supernatant prior to pre-enrichment.

kind regards

ketil

CD63 and couple other options are widely used by the community. but they are not perfect. we all hope to agree on exosome/EV nomenclature and markers fairly soon, but its not trivial. at the moment the easiest way seems to have size-based definitions of nano/micro-vesicles

Dear all,

actually I isolated exosomes form  FaO rat hepatoma cell lines,

confirmed exoxomes presence and diameter by electron microscopy.

Then I analysed by WB C63 or CD9 and no band could be detected.

I analysed 3 ug of tot proteins, do you think that is too low protein quantity or I should try with another marker?

Grazie

Elena