I am trying to purify the exosome from biological fluid. Most protocols say to use Sepharose CL-2B or 4B columns for purification of the exsosome. I was just wondering if it would be okay to substitute Sephadex instead of Sepharose CL-2B as we don't have the medium in the lab? Also many protocols use SEC after ultracentrifugation or cushion centrifuge. Would it be okay to use SEC after precipitation by Kit and achieve similar purification?
Hey Razieh,
If you're interested in surface-marker specific selection of exosomes using an immunoaffinity-chromatography based approach, you might want to try Fab-TACS® Exosome Isolation Kits (you can find all the information about the technology here: https://www.iba-lifesciences.com/exosome-isolation.html). The reagents are reversible, this means that the exosomes are label-free after the isolation. The columns have a high capacity and are also suitable for biofluids. The protocol is very fast and you don't need ultracentrifugation.
I attach some application notes that show exosome isolations from different sample types.
The CL in Sepharose CL-2B stands for cross-linked, this gives the beads greater mechanical stability against the back-pressure of the column, it also allows SEC in organic solvents. In addition, the cross-linking allows charged groups present in natural agarose to be removed during manufacture, which otherwise would have an IEC-like effect.
The number (2, 4 or 6) is the percentage of agarose in the gel. Higher percentage makes the gel more stable and reduces the pore size. Given that you want to separate large particles, you want a low agarose percentage (which makes cross-linking more important).
SEC is a non-binding technique and requires small sample volumes (max 1% of column volume, better less) to give good resolution. The technique is therefore unsuitable to capture the desired product from large volumes of biological fluids. In the procedures you mentioned, this is the job of ultracentrifugation, Alexandra Nordlohne mentioned an affinity matrix that can also do that job.
A good source for info on SEC is the old Pharmacia booklet now available under https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/General_Information/1/ge-gel-filtration.pdf
Thank you very much for your helpful suggestion. I read the Pharmacia booklet and the picture that Engelburt and Thijes suggested. I figure out that the biggest pore size for Sephadex resin is 600 kDa (for Sephadex G200) while in the article with DOI: 10.1038/s41598-017-10646-x, the authors said that the EVs are larger than 700 kDa. I am not sure that I truly conclude that if I use Sephadex resin for purification, I will lose a lot of EVs especially after centrifugation because of vesicle aggregation.
Yes, you can use SEC after exosome isolated by precipitation method. Please find the attached file. I hope this will help you.
Thank you for sharing the Quantitative proteomic analysis of extracellular vesicle subgroups isolated by an optimized method combining polymer-based precipitation and size exclusion chromatography (2021) reference. Very helpful.
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