I isolated a large amount (I hope) of exosomes and I need to quantify the membrane proteins with a Bradford assay before doing a WB. I started off with 70 ml of conditioned medium which I managed to concentrate to about 1 ml before ultracentrifugation. After ultracentrifugation I added 200 ul PBS to my samples. My expectation is that the concentration of exosomes is quite high. So now I need to do a Bradford assay and I would like to know how to dilute my samples.
Hi Michal
Why do not try directly reading the absorbance at 280 nm in a Nanodrop? It will give you an estimation ot the protein content of your sample as accurate (or inaccurate) as Bradford, but less time consuming, and you can try several dilutions ....
Hi Michal
Certainly, nanodrop is a very good option but if you dont have it, you can try a colorimetric assay. The Bradford method has a linear response until approx 300 micrograms/ml. The BCA method has a more extensive range (2mg/ml) so, probably you can do less dilutions to quantify your protein. I suggest you use the BCA method.
Good luck,
Hi Michal,
We use Bradford for exosomes, but because that method does not disrupt the exosome membrane integrity one will not get a fully representative quantitation of your exosomal protein, so expect a lower value that if you were to measure protein with a lysing technique.
Hi Michal,
I've used Lowry assay to quantify exosomes' protein content.
On the other hand, can you explain me how you concentrate your medium before centrifugation?
Hello Michal,
As Phillip has explained already, you certainly need detergents to dissolve your samples. Besides a non-ionic detergent like TritonX-100 some SDS (only low concentration, e.g. 0.2%) should be included in the sample buffer as well. A standard recepy is RIPA buffer, PBS alone will not work.
Then the next question is:
Does is matter much what lysis buffer I use? I've used RIPA before but I see in publications that tergitol-type NP-40 is used too. Which is better?
@ Dirk Breitkreutz
Hi Dirk. How long do you incubate your exosome sample in RIPA lysis buffer for before you do your BCA or bradford assay?
I have been getting a very low protein concentration from my exosome sample in PBS and maybe I should lyse them before doing protein quantification
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