I have isolated extracellular vesicles (EVs) using PEG-based precipitation and removed PEG using a 50 kDa filtration step. I now want to quantify the protein concentration of the isolated EVs. Most of the literature I've reviewed reports using the BCA assay, often with protocols involving lysis of the EV membrane. However, I am currently unable to perform the BCA assay. While a few studies mention using the Bradford assay, I couldn't find a clear or standardized protocol for exosomal protein quantification using this method.
Could anyone please share a reliable Bradford protocol specifically for EV protein quantification, or suggest alternative suitable methods for estimating EV protein concentration without relying on BCA?"
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I have used Bradford for this in the past. Lyse an aliquot of EVs with RIPA buffer on ice for 30 minutes. test various dilutions of lysate to ensure you are on the standard curve. I used 1:5, 1:10, 1:25, and 1:50
Thank you soo much for the valuable answer maam.
I actually want to correlate the protein concentration with the concentration of EVs, but I am still wondering if when measuring the protein concentration by Bradford's Method, in order to correlate it with the concentration of EVs, do the proteins have to be previously extracted from the EVs or do I just add Bradford's reagent on my sample with intact EVs without any protein extraction?
If you do not extract/lyse the EVs then the protein estimation will not be accurate. you could try testing unlysed EVs alongside and compare but it will likely come out very low. You can then correlate the lysed protein concentration with the concentration of particles in particles per ug of protein. see literature on this as a quality measure PMID: 24009896
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