Hi all,
I have been trying to measure a concentration of my exosomes from adipose-derived stem cells. I used 700 ml of conditioned media to isolate exosomes however, when I did BCA assay it gives me a very low concentration of protein (0.25 ug/ul). This is very strange because I did even see some pellet at the end of the isolation step.
I wonder if the low concentration from BCA assay is because I did not lyse my exosomes with lysis buffer using such as RIPA buffer.
Does anyone have a good experience measuring concentrations of exosomes with BCA assay?
Johnathon Anderson
Hi Johnathon,
Thanks for your reply.
I get my exosomes pellet by ultracentrifugation, and I reconstituted my exosomes with PBS as I need to use them for my downstream assays.
Can I directly add RIPA lysis buffer to exosome suspension?
How much lysis buffer do you usually add? Is it 1:1 volume ratio?
Could you please give me a protocol for lysing EVs?
According to your description, the lipid volume is quite large. our lab also do some research about exosome, mostly, 300ml lipid is enough, the protein concentration about 20 ug/ul. maybe you should consider the experimental process if exist any error.
I agree with Johnathon: lyse and use micro-BCA. Note that, generally, the more pure your EVs, the higher the particle:protein ratio. Different EV separation methods may have drastically different levels of non-EV protein co-isolates.
Dear Yinan,
from my laboratory practice I observe that I cannot expect high concentrations of protein in contditioned media. To use EVs for proteomic research we concentrate media to obtain EV enriched fractions, usually by filtration methods, then centrifugation. As Keneneth said, calculate particle:protein ratio, it is a good standardization protocol.
I am working with peripheral blood samples from pregnant women, and I am trying to isolate microvesicles just with ultracentrifugation. We collected the samples on citrate of sodium, and then we centrifuged it at 400g 20min to obtain the plasma. Next, we diluted the plasma with PBS (1:2) and centrifuged at 1500g 30min to discard apoptotic bodies. The supernant was then centrifuged at 15000g 30 min to obtain microvesicles. Finally, we mad one more centrifugation at 15000g 30 min to wash the microvesicles.
In general, it is said that at the last centrifugation I should have seen a pellet, but I almost could see it. Apart from that, I had a very low efficiency as it was evidenced by quantifying proteins with Bradford.
Can anyone give me an advice to perform the protocol?
Besides the protocol´s problems I have to choose three antibodies to characterize the microvesicles isolated. I was thinking in CD81, CD63 or CD9 because they are not only specific for exosome. Anyone can give me others options, or the better biomarkers for them?
Thanks!!!!
Certainly, lysing the exosomes with RIPA (1:1) and water sonicator for 10 min increases the readout of protein concentration with BCA assay. In my experience, lysing exosomes increases 20-30% the amount of protein detected. If your protein concentration is more than 0.125 ug/ul of exosomal protein, you are in the quantification range of BCA. We usually get 60ug of exosomal protein from that amount of conditioned media using HEK293 cells, and after a sucrose gradient. We do not trust direct pellets from conditioned media, since there are other things apart from exosomes in that pellet, that's why we perform the sucrose gradient. Using a bioreactor might give you a better exosomal yield, but it is less cost effective.
Kenneth W Witwer hello
which lysis solution do you use with microBCA assay please?
Thank you
Have you tried to pre-concentrate your media by ultrafiltration? it might help
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