I'm collecting exosomes from condition media for proteomics purposes. Is adding the extra step of sucrose gradient UC essential for getting an acceptably pure sample? If so, what protocol do you recommend? Thanks in advance
Ultracentrifugation with sucrose gradient provides very clean exosome fraction. The protocol is lengthy and tricky though. You might consider commercial reagents such as Total exosome isolation (from cell culture media), which enables fast and simple recovery of exosomes
It depends how pure you need your exosomes to be. Ultracentrifugation alone will always result in protein contamination. Sucrose gradient centrifugation is a good way to separate exosomes from other vesicles but HDL contamination of the exosome fraction can result as the density of exosomes and HDL is the same. Size exclusion chromatography, affinity chromatography and magnetic bead separation also produce very pure exosomes.
The subject is covered in depth in the ISEV position paper (attached)
sucrose density gradient following ultracentrifugation is the well established method to isolate pure population. Else you can try magnetic separation using exo specific antibodies in order to isolate sub-populations of exosomes. Check products.
Sucrose gradient ultracentrifugation would be a better option for exosome isolation. If you perform only ultracentrifugation, then you can wash the pellet several times with PBS to remove protein contamination.
It really depends on your tissue/cells of interest, some methods which are working perfectly to isolate exosomes from Urine, would not work as good for plasma or blood cell cultures. But in general and as others also suggested, you may follow the general protocols using ultracentrifugation following Sucrose gradient and try to optimise it based on your own experience. The ISEV reference by Chris can be very helpful!
The problem is defining an acceptable pure sample. do you consider a fraction with other extracllular vesicles such as microvesicles also a pure isolation of exosomes?
Thanks all for the helpful replies! The ISEV position paper was very useful.
Arian, the level of purity that I need is minimum contamination with both extracellular vesicles and protein aggregates, etc. Since I don't want to exclude any subpopulation of exosomes by using magnetic beads, seems like sucrose gradient UC is the best to choose.
Please note that we have recently quantified the recovery and enrichment (compared to proteins and lipoproteins) of vesicles isolated by size exclusion chromatography with a CL-2B column. http://www.journalofextracellularvesicles.net/index.php/jev/article/view/23430
Another good paper on this subject was published last week
http://www.journalofextracellularvesicles.net/index.php/jev/article/view/24858/35786
Hi
I want to kown the ISEV position paper ,can you give me a send.Thank you very much
Haihua, Check out the second comment to this question by Chris Gardiner. There is a link...
Thank u very much，but this paper do not provide the exact protocol of UC following sucrose cushion ,can u offer me a protocol to isolation serum exosomes in the method of UC following sucrose cushion.I will very appreciate ,thank u very much
Hi,
for direct capture of exosomes using Dynabeads and how to optimize for flow and western please see our webinar which can be downloaded from my profle - or get in touch with me.
kind regards
ketil
Hi,
we will now very soon also publish a bit more around direct capture of exosomes and which factors that should be addressed.
kind regards
ketil
try the Total exosome isolation reagent (from cell media) - it enables fast isolation of highly clean exosomes, comparable to ultracentrifugation protocols. in higher yields.
Any effective protocol based on PEG precipitation for isolation of exosomes from urine
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