I isolated EVs from iPSC-derived NSC lines using a commercial kit and characterized the Evs for presence of classical EV positive Markers and detected the expression of CD63, TSG101, and Calnexin (ER marker) as a negative marker. However, when I did the BCA protein quantification assay, protein concentrations were very low in EVs, while the house keeping genes; Actin and GAPDH were also not expressed in Evs but present in the parental cell lysates (image attached). Even though there have been inconsistencies in published EV studies on whether the “house keeping genes“ should be present in Evs or not. I would like to know the opinion of experts in this area, thank you.
Dear Ayodeji Abijo,
EVs have a distinct protein profile rather than a WCL. They are enriched in specific markers such as: tetraspanins (e.g., CD9, CD63, CD81), Alix, TSG101. These proteins are commonly used as positive controls for EV identification. The absence of Actin and GAPDH can also indicate that your EV preparation is relatively free from cellular debris or lysate contamination, which is a good sign for the purity of your EV sample. So housekeeping genes will be absent in most cases.
Best
Subhajit
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