I isolated extracellular vesicles from iPSCs-derived Neural stem cells via a combination of ultracentrifugation with a commercial kit and characterized the EVs using NTA to determine the size distribution and concentration of the EVs. Western blotting was used to determine the expression of the tetraspanins (CD9, CD63 and CD81). Consistent across several experiments, is the expression of CD63, and another EV marker (TSG101) With Calnexin as a EV negative marker. I noticed that there is batch to batch variability in the expression of CD9 and CD81. While the known molecular weight of CD81 is ~22-26kDa, I detected mine above 55kDa (image attached). What could be responsible for this? Can CD63,TSG101 and the Calnexin (negative EV marker) without CD9 and CD 81 expression pass for EV characterization?

Experts opinion needed.