Hello everybody,
I am currently working on EV isolation and caracterization and exosomes by ultracentrifugation. I used beta actin as a loading control for my blots but i don't really know if it is correct or what do you recommend me to use? Actin was also present in my exosomal fraction which is supposed to be at 100k speed. But i have difficulties to see exosomal markers such as CD9 and CD63 and Alix from abcam.....so i don't know am I supposed to see actin in exosomes or is it cell contamination?
Besides, which antibody and which company do you recommend me to buy antibodies for exosomes markers from?
Thank you so much
Hi Lydia Amari CD9 and CD63 are the right marker to detect exosome. The antibodies from Abcam are fine. if you don't see the these markers and your Western blot is OK, either you don't have exosome in your sample or you not enough of material after UC
For exosome composition you can also have a look at this paper
https://www.cell.com/cell/pdf/S0092-8674(19)30212-0.pdf
Hello Nguyen,
Thank you for your answer. Yes the Red ponceau seems good protéines are here. But still i used abcam antibodies but no exosomes markers visible....
Thank you so much
Hi Lydia, CD63 and TSG101 should work for WB analysis, but you should keep in mind some small vesicle heterogeneity by several purification procedures (commercial kits, gradient centrifugation...). Then, differential surface markers might be present. For more references you could look at https://exosome-rna.com/rna-in-extracellular-vesicles/.
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