Hi,
I am having trouble isolating microvesicles from 1ml of healthy patient plasma using size exclusion chromatography. I am trying to validate this method using western blot with CD63 and CD81. Vesicles are isolated according to this protocol: http://www.journalofextracellularvesicles.net/index.php/jev/article/view/23430 . One small difference, I use ammoniumbicarbonate instead of pbs+citrate.
I have isolated 26 fractions and tried western blotting fractions 8 till 11, based on previous results with Concentrated cell secretome which showed perfect results. However when I use the same method for plasma, I get no results. I have tried concentrating the 0.5ml fraction using: centrifugal evaporation(worked perfect for secretome), TCA precipitation, ultrafiltration using Amicon 3k cutoff filters.
Where is it going wrong, is the input volume too low? Is it the antigene that is somehow cleaved in plasma?
chromatography on large columns is not a good option- as you are diluting your samples. ideally you want clean exosomes/EV, but also in a concentrated form. Its tough, I also looked into this... Try the Total exosome isolation reagents (5 options for misc fluids + CM)
Tim,
I'm using SEC and comparing to ultrafiltration and Exoquick.
Can you please tell me which TCA precipitation protocol did you follow? And if it worked or not?
Thank you. Best regards.
Dear Vanessa,
what finally worked for me was using ultrafiltration (300k cutoff) and 1:150 antibody dilution for CD63.
Tim
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