I isolate exosomes by differential centrifugation, I use protocol Théry et al. (basic protocol 1). (I use pre-spin FBS in cell culture medium.) When we perform electron microscopy there is a lot of protein aggregates. So we tried to purificate exosomes by 30% sucrose cushion, but there are still some visible aggregates in electron microscopy pictures. Do you have any suggestion how to separate these aggregates from exosomes? What could these aggregates be? I found immunocomplexes in the literature, but something more concrete… albumin from FBS? Do you face the same problem? Thanks a lot in advance!
If you are purifying the exosomes from cells where some proteins have been overexpressed, you are likely to find a lot of exosomes and there is no way of avoiding it, as far as I can tell and as far as I have tried. However, you can remove the exosomes from your preparation using CD45 depletion. CD45 is specifically incorporated into exosomes and if you remove these CD45 containing moieties, you could get higher purity for your prep. ( David E Ott, Rev. Med. Virol., 2008; 18: 159-175)
Agree with Sanath, one way is to use some "specific" antbody against exosomes, bind these to magnetic beads (or buy premade anti-CD63 magnetic beads) and wash away before elution. But it all depends on the downstream work...
-you might also go into preparative field flow fractionation?
I have the same problem. with my preparations.. I am planning to use a sucrose gradient, hoping the protein aggregates will sediment in other fractions than the majority of exosomes... Do you think it is a realistic option?
@Laurent Galio,
I have been purifying HIV-1 virus preparations by pelleting through a 20% sucrose cushion, and I still find impurities, which I like to term exosomes or vesicles- I have not done enough experiments to confirm what they are.
But in over expression experiments, due to protein misfolding or just plain over expression, proteins are present all over and the cell tries to get rid of excesses by whatever means it can. So, it will produce exosomal vesicles packed with proteins, which can be pelleted along with other protein rich vesicles or virus particles.
Added to these, over expression of glycoproteins and transmembrane proteins always leads to some toxic side effects to the cell, one of which is increased membrane blebbing and exosome production. These exosomes could very well be packed with proteins that would allow them to pellet along with virions.
Adding excess buffer to your sample and re-concentrating the sample using a 100 kDa MWCO Amicon can eliminate many of the contaminant proteins. Basically, it's just washing your exosomes.
Thank you all! Yes, antibody is probably the best method how to increase purity of isolated exosomes. I would like to study different fractions of exosomes proteins or complexes separated by chromatography, so i need a high yield of exosomes and this would be costly.
@Laurent Galio: Yes, I think it is realistic option, it is used in a lot of articles and maybe it is even better than sucrose cushion. But I think that preparation of sucrose gradient is more complicated. Is there any trick for gradient preparation?
EM protocols are tricky, there are many things that could go wrong. But if these aggregates are of protein origin- they can be eliminated by a brief proteinase K teratment (and exosomes will stay intact)
We observed large aggregates via SEM when we freeze-thawed our exosome preparations prior to preping them for SEM. By switching to 4C storage instead of freezing the exosomes the levels of aggregation were largely resolved.
We have resolved how to disperse MCVs and its all about the buffers used. We have a buffer Vindicrin that is utilized to disperse EVs and allow for good antigen (CD63, 14-3-3 etc) presentation that are not seen in PBS. Contact me if more details are needed but protien aggregates are trapped in all methods of exosome isolation. DIsperse treat with protenaase K and then lyse otherwise a lot of IGG, lipoprotein, high abundant protein and RNA material are associated with the seroproeinaceous aggregates < 100 nM that we call exosomes.
I have recently also seen protein aggregates in my exosome preparations and I am wondering about trying optiprep. Did you finally manage to separate your exosomes from the protein aggregates?
I changed isolation technique and I used magnetic beads with CD63 antibody and it looked much better but now I stop isolation of exosomes for a while.
for challenging samples such as plasma, pre-treatment with proteinase K helps to deplete the bulk of protein. conditions shoudl be carefully optimized though if you are interested in analysis of exosomal surface proteins (which can be partially chewed)
- Badges
- Science method