With exosomes isolated from human umbilical cord cells I keep seeing the same high flourescence band in at the same molecular weight 58Kda on a 10% SDS-page gel. This is across different exosome preps which have been lysed in RIPA buffer. I use FBS which has been ultra-centrifuged to remove bovine exosomes but could the unidentified band from contaminants from this FBS?
Hi Emma
It may be Albumin (around 65 kDa). Please see the following link;
https://www.researchgate.net/post/Why_am_I_not_seeing_banding_on_exosome_western_blot_for_Alix_Flot1_TSG101
If you can, try comparing your protein concentration to others that use the same cell type. Then you could have a hint toward whether there may be protein contaminants.
What were the conditions of the FBS centrifugation ?
All the best
Jason
Agree with Jason. We have performed HPLC on many ultracentrifuged samples of culture medium (FBS exosome depleted) and albumin is a prominent contaminant (~60 KD) necessitating multiple washings, or sucrose gradient, or elimination of contaminant proteins by size exclusion HPLC (although significant loss of exosomes depending on system).
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