Is there anyone isolating exosomes from mouse embryonic stem cell ?
How much proteins would you expect after ultracentrifuge ? The protein yield I got was extremely low, sometimes it's no more than 0.1mg/ml ..
Is this normal or just me?
Anyone got experience to share ? THANKS !
Hi,
I was isolating extracellular vesicles from stress-exposed epithelial cells and the average protein concentration in the pellet was of 0.2-0.3 mg/mL from ultracentrifuging 3 mL of cell-conditioned medium. It should be mentioned that the pellets contained exosomes, microparticles and apoptotic bodies altogether.
Therefore, it might be that your yield is good enough (depending on the amount of media you use to isolate that 0.1 mg/mL of protein).
Thanks a lot Alexandra , but this 0.1mg/ml is the contraction of 200ul exosomes isolated from like 30 to 60 ml of conditioned medium . so it's really little comparing to your yield .
I thought maybe I lost most of exosomes when I discard the supernatant after centrifugation , how do you usually remove the supernatant ?
Those yields are fine given the small culture volume.
When we isolate dendritic cell exosomes (high producer of exosomes) we will get 1-3 mg/mL of exosomes in about 200uL by UC. But, this comes from 0.5-2L of culture medium. You'll get much better yields by ultra-filtration, but will likely need LC cleanup to remove any protein contaminants.
Hi again,
I usually pipett-out the supernatant. But, as I mentioned before in my pellet there are also other types of extracellular vesicles and that can explain the higher protein concentration. Exosomes are the least abundant (if I were to judge from the immunoblotting for surface markers), so I still think you have a reasonable yield.
- Badges
- Science method