I'm having trouble in immunoblotting(western) CD63 especially in exosomal proteins, since the antibody works well in non reducing conditions. My proteins were in RLN buffer. Is this going to affect CD63 expression. The protein concentration was about 0.5 ug/ul.
Can be a lot of reasons. Are you sure that you didn't loose your exosomes and that the concentration is good?
Yes, I am. Becoz half of the exosomes from thesame extract has been used for RT-pcr and they look good.
I would guess :
either concetration is too low for detection in wb
or you cells don't produce a lot of cd63
or cd63 is degradaded an the epito is not longer available
I would try to use other antibodies like alix or tsg101 and see if you have the same problem.
hi, which anti-CD63 clone is used for labeling? The TS63 usually works well (non-red conditions) for detection of CD63 in exosome lysates. can other exosomal markers be detected? CD81, CD9?
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