Hi all,
I was doing a western blot with my exosome samples, and I found the presence of Calnexin in them. Now, I know technically it is not supposed to be in there, since Calnexin is a negative marker of exosomes, so what does this mean? In case it helps, I extracted my exosomes from HUVECs cell culture media.
Thanks,
Shalini
Shalini,
What criteria did you use to validate that your exosome preps are clean? ...that they are not contaminated with other EVs? ...or even don't contain proteinaceous aggregates? ...or that calnexin is not a 'sticky passenger/stowaway' in your prep?
In other words how confident can you be that you actually have a clean exosome prep? When you see results like this, the first question you ask is "what did I do wrong?" I am not saying you are wrong; you might not be wrong! This might be real, but you need to eliminate all other possible explanations before concluding this!
D.
Forgot to mention... How confident are you that your Ab to Calnexin is clean? ...that it does not cross-react with other targets?
@David Hi David, I checked the size range and structure via TEM imaging..those are definitely exosomes...but even if we consider there are other EVs, it should still test negative for Calnexin since Calnexin is negative marker for EVs in general... The Calnexin primary antibody has been used 2-3 times though..but I think they're pretty clean..
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