Hello!

Right now I am struggling to perform a western blot with plasma-derived human exosomes. I want to confirm the presence of typical exosomal markers, e.g. TSG101, CD63 and CD9.

I know that I obtain exosomes from human plasma (using SEC columns) by looking at them in electron microscopy.

Specifically, we first isolate the exosomes using HansaBioMed SEC columns and then perform post-filtration with 300kDa and 10kDA filters to purify and concentrate the sample.

When I try to do the WB for my plasma exosomes something strange happens: I don't see any bands for CD9, CD81 and TSG101, but we see a good signal for CALNEXIN, which ironically is our negative control.

We have tried adding more starting protein, using reducing and non-reducing conditions and nothing. Please find attached two of the protocols used in case they are useful.

Has anyone experienced similar issues and has some advice?

Thank you very much in advance!

Kind regards,

Paula