Hello!
Right now I am struggling to perform Western Blot with human, plasma-derived exosomes. I want to confirm the presence of typical, exosomal markers - e.g. Alix, CD63 and CD9. I know that I get exosomes from human plasma (using the Total Exosome Isolation Kit from Invitrogen) by seeing them in electron microscopy and NTA.
I also confirmed that the antibodies are working by checking them with cell culture exosomes (from HCT116 and LIM1215 cell) - for those samples I see a nice signal in all antibodies.
However, when I try do to the same for my plasma-exosomes - I don't see any band at all. I tried to increase the volume of plasma starting material (500 µL - up to 1 mL), tried nitrocellulose and PDVF membranes, removal of the junk plasma protein before exosome isolation - but nothing seems to work.
Has anyone experienced similar issues and has some advice?
Thank you very much in advance!
Lena
hello Lena Bohaumilitzky may I ask what are you blocking the membrane with? I assume you are doing denaturing WB. It is possible your exosomes are not lysing properly to expose the target. In vitro exosome fraction is alway cleaner and concentrated while human samples' exosomes tend to be heavily coated, hetrigenous and require rigorous lysis. After invitrogen precipitation step are you lysing the pellet or resuspending it in RIPA buffer? Are determining the protein concentration or loading by volume? It is best to quantitate. Check this reference (https://mdpi-res.com/d_attachment/bioengineering/bioengineering-06-00008/article_deploy/bioengineering-06-00008.pdf) to trouble shoot your protocol. Good luck.
I usually use 10% SDS-containing lysis buffer (supplemented with protease inhibitor cocktail etc., like regular lysis buffer) to lyse the isolated exosomes. Sonicate well to break down the exosomes. Here is some example protocol you can briefly lookup:
https://www.stemcell.com/how-to-characterize-extracellular-vesicles-by-western-blotting.html
Hi Rabeah Al-Temaimi
I tried 5% milk and 5% BSA in TBST, did not make a big difference for me. I am lysing in 1x RIPA buffer with protease inhibitor cocktail (also tried with and without sonication) - I get protein in the Bradford assay and normally load 20-25µg of protein on my SDS-page.
And thank you for the paper! It just confuses me more - as they get a band for the exosomal markers with the Invitrogen Kit (without proteinase K treatment) I don't get that at all...
Best,
Lena
Lena Bohaumilitzky I understand your frustration. Try perhaps to prepare a fresh RIPA. It is possible that your sample needs boiling in urea? I had that issue with mito transmembrane proteins, that were nicely detected in cell culture. sincerely wish u good luck.
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