Hi, I am doing western blotting of isolated extracellular vesicles, and I have albumin and calnexin contamination that I don't know why. Is there any way to avoid it?
Hi Carmen María Picazo Córdoba,
How do you isolate your EVs? Since you got Calnexin (ER marker oftenly used as a control) in your EV preparation i guess that something went wrong during the isolation process.
I´ve tried ultracentrifugation and SEC (chromatography exclusion size) methods, and with both I get the calnexin and bsa bands when I do the western blot. I don´t know what I´m doing wrong. Any ideas??
If your doing in vitro cell culture, Albumine will originate from the serum of your culture media. It does contain vesicles as well so first I would advise you to prepare ev-depleted media unless you already do that.
In both methods, it is important to follow the first steps of centrifugation with low rotation force (300g for cells, 2000g for apoptotic bodies. Those two steps are common in UC and SEC) to avoid dead cell breakage that will contaminate your ev pellet. Do you follow those steps before 100k UC or SEC columns?
You should at least get rid of calnexin this way, for Albumin it could be more complex to completely remove it i guess, even with exo-free media. I would recommend you to use SEC instead of UC (depending on your assay) since it will purify your EV fraction from the soluble proteins and very small aggregates.
Are you using your culture media supplemented with FBS-depleted serum or FBS?
If it is the latter, then the probability of albumin exists, but you would see it much in the late fractions if you use SEC.
Try using FBS-depleted media to confirm if other contaminants exist.
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