I am trying different methods of isolation of exosomes to validate the most convenient one for further studies.
I have used the protocol for precipitation with PEG (30%) and now I am considering to do size-exclusion chromatography with Sephadex CL-4B.
My question is following: Is it necessary to remove PEG (if yes, is it better to perform some washing step or ultracentrifuge) or I can load the sample as it in columns?
Thank You!
PEG depending on length and percentage, and amount remaining with your exosomes- might have negative downstream effects. So spin down your sample couple times, carefully remove all droplets from the tube wall, so that only exosome pellet remains, and resuspend it in PBS or other buffer. Gel filtration with optimized resin will allow to deplete remaining PEG (if eg you want to have highly clean exo sample for transfectons), but this will result in substantial sample dilution, for longer columns.
Hello Berina,
As described in Montaner-Tarbes, 2016 Sergio Montaner-Tarbes , you can resuspend your Exo-PEG pellet in PBS and load it onto a SEC column (Article Serum-derived exosomes from non-viremic animals previously e...
). As Alexander said, it can give you high volume of samples, so the sepharose volume you need, would be larger as mentioned in the mentioned work.Ana
Thanks Ana Gámez-Valero for the suggestion to Berina Šabanović . We did that and worked perfectly to get rid off the PEG in the EVs suspension. PEG precipitation is a technique applied also to precipitation of infectious viral particles with concomitant separation by size exclusion chromatography. Retroviruses remain infectious after this process as a prove of maintaining biological functions after enrichment and purification. If you need anything else, just ask :)
Cheers,
Sergio
Ana Gámez-Valero , Sergio Montaner-Tarbes , Alexander Sasha Vlassov thank You very much for Your answers!