We are trying to isolate CD63+ exosomes in order to characterize them by electronic microscopy, Nanosight and ultimately by proteomics/transcriptomics. To isolate them we use the “Total Exosome Isolation” kit (Invitrogen cat#4478359) obtaining the total exosome population from our cell’s culture media followed by and immunoisolation of the CD63+ exosomes subpopulation using the Exosome – Human CD63 Isolation/Detection kit (Invitrogen cat#10606D). This second kit consists on magnetic beads coated with anti-CD63 antibodies. Our main concern before analysing the particles that we obtain is detaching the exosomes from the beads, especially for Nanosight evaluation. The techsupport team from Invitrogen could not recommend us any buffer but their suggestion was using a solution with a high salt concentration or low pH. Still, they are not sure it will work.
Can anyone suggest us a buffer or strategy to detach the exosomes from the beads’ surface?
see http://www.nature.com/articles/srep33935#f6
Hope that helps. At least the paper itself is really worth reading for EV-related technologies. I would highly recommend it. Philippe
For dissociation of antigen-Ab interactions, two methods are commonly employed: use of low pH buffer (such as Glycine buffer pH 3.0-3.5) and high salt. Both methods work adequately well for relatively stable ligand and antibody (depending on what you are purifying, one may be sacrificed if necessary).
If you choose salt, rather than NaCl, use 2.5 MgCl2 at neutral pH - it is tolerated better than other options.
If possible, try an ultracentrifugation based method to avoid the problem of dissociating interactions by harsh methods.
1. íf you know the ab's epitiop, you can try elute by excess epitop. however, this method was used for small chemical epitops mostly, i think. (nowadays, it is relatively easy to generate rec proteins. however, you should know the epitope of your ab well, if it is glycosylated, method in E coli will not work. According to CD63 biology (just a brief check), this approach does not look promising.
2. you can try to release from beads by disrupting the binding abs with a reducing agent (DTT, mercaptoethanol) similarly like stripping a blot. it can affect the vesicles probably..
Hi,
When developing the Dynabeads CD63 beads for exosome isolation and analysis the main focus was to offer a solid support platform for exosomes which could be recognized by the flow scatter and then be able to do detailed analysis of stained exosomes located on the bead surface . The Ab-Ag interaction in this case is very strong and no release mechanism was therefore included in this product. The low pH or high salt strategy can of course be tested but it is most likely not very effective.
kind regards
ketil
Thank you for your suggestions guys, and sorry for my late answer. We have been trying 2 different home-made buffers:
- 1% formic acid in PBS
- 0.2M glycine, Tris-HCl pH 2.8 in PBS
Any of them worked in our hands. So finally we purchased EXOFLOWBUFR-2 from Cambridge Biosciences and is working fine, according to Nanosight and ELISA results.
Hi Victor,
Elution of exosomes off of magnetic beads is common practice. I would reccomend against a pH-based elution as it can be difficult to regain neutral pH and exosomes can lose some stability when left at pH < 3 for an extended period of time.
I would recommend either a salt-based elution or using the elution buffer (ExoFlowBuffer2) found in SBI's exosome capture kit (Exo-Flow/IP). There are a variety of different biotinylated antibodies (CD63, CD9, Rab5b, etc.) provided in the ExoFlow kits that can serve your purpose
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