I'm trying to use microBCA por protein estimation for exosome samples. Does anyone have a optimazed protocol that can be applied? I'm trying to reduce volume os standars and samples but the assay is not working.
Hi Stepano,
-Before you consider there is anything wrong with the microBCA kit, try to quantify cell lysate first to make sure the BCA reagents are not off.
If you tried that already , the your exosome isolation method may have an impact on BCA quantification. I used to isolate exosomes with a comically available kit ( I can't name the manufacture that would be bad) but it didn't work well for me. One of the reagents from the exosome isolation interacts with some of the BCA reagents, giving false positive signals on BCA readouts. And the yield was extremely low. I think some of the commercial kit has limitations on how much exosomes you can isolate. Once you exceed the capacity of the isolation kit, you won't be getting any more exosoms because the kit becomes your rate limiting factor.
I switched to ultracentrifugation for exosome isolation, ever since then the BCA worked wonder for me. Hope this piece of information helps. Good luck.
I agree with Tommy, some of the kits and isolation procedures influence the assay, I had trouble with exosomes isolated with Iodixanol Gradients and proteins purified trough Trizol LS. BCA was just showing high levels of proteins where there were no proteins. Make sure the buffer you use is the same you use for BSA standards and that this one is compatible with the BCA. Besides that, such kits works good enough in my experience when you precipitate your exosomes using Trichloro acetitic acid and suspend the proteins in BCA compatible buffer. You might need to precise what is not working exactly. Is the standard curve odd ? Is it too high or too low ?
Abderrahim Benmoussa, Brother. i'm facing a problem similar to this question. i precipitate the Exosome pelleted proteins with Acetone (absolute). But after precipitation i see that the protein pellet is very sticky and i am not able to dissolve it in 6M Guanidine hydrochloride. I also think that it is not compatible with the BCA and the reading from BCA is not true. Can you please guide me, what is better option to dissolve the Exosome pelleted proteins and also what is best method to measure the protein concentration.
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