I am working with peripheral blood samples from pregnant women, and I am trying to isolate microvesicles just with ultracentrifugation. We collected the samples on citrate of sodium, and then we centrifuged it at 400g 20min to obtain the plasma. Next, we diluted the plasma with PBS (1:2) and centrifuged at 1500g 30min to discard apoptotic bodies. The supernant was then centrifuged at 15000g 30 min to obtain microvesicles. Finally, we mad one more centrifugation at 15000g 30 min to wash the microvesicles.
In general, it is said that at the last centrifugation I should have seen a pellet, but I almost could see it. Apart from that, I had a very low efficiency as it was evidenced by quantifying proteins with Bradford.
Can anyone give me an advice to perform the protocol?
Besides the protocol´s problems I have to choose three antibodies to characterize the microvesicles isolated. I was thinking in CD81, CD63 or CD9 because they are not only specific for exosome. Anyone can give me others options, or the better biomarkers for them?
Thanks!!!!
Hello Fiorella,
Could you please confirm what was the speed of your last centrifugation? 15,000g is not enough to pellet exosomes .
Hello Daria,
The last centrifugation was at 15,000g to pellet de microvesicles. I do not want to obtein exosomes, that´s why I do not go on with the 1000,000 g centrifugation.
Hi Fiorella,
I am not familiar with the ultracentrifugation protocol but if you have issues with your exosome isolation protocol, there is other methods available :). Cell Guidance Systems combines precipitation and size exclusion chromatography for highly pure exosome samples. If you are working on high volumes (1 ml), you can add your sample directly on the top of the column. If you want to have a look, please click here:
https://www.cellgs.com/products/exo-spinand8482.html
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