I've been using microBCA for protein concentration estimation for exosome samples. I used to get very consistent results with lysed exosome samples, however this is no longer the case and I am getting very low, and even negative protein concentrations calculated using this assay now.
I have thought it may have been something wrong with the lysis buffer, I normally used a 1% NP-40 lysis buffer, and have tried using RIPA buffer as well as a 0.1% SDS buffer. I've checked for incompatibilities with the reagents and have ruled these out.
I have also used a Bradford assay on the same samples, and instead of low and negative values, get uniformly high values, but when these samples are run on a gel, I see nothing appear using Ponceau-S or Coomassie staining suggesting there's nothing there.
I'm at a loss and any help or advice you could offer would be appreciated!
Just checking that you are using NP-40 and have not been given Nonidet P-40 or IGEPAL CA-630 instead? As they are often called NP-40 but are a completely different type of detergent and won't be strong enough to achieve what you need.
Hi Stanley, the NP-40 is in fact Nonidet P-40 (in my old 1% NP-40 lysis buffer) and is IGEPAL in the RIPA buffer I've begun using. Why would this not be strong enough for exosome lysis? Should I be using Triton X-100 instead?
Thanks
Assuming that your protein standard curve looks good (does it?), you can spike a couple of your exosome samples with protein standard and re-measure. Depending on how it looks, you might need to consider some kind of clean up before assaying, e.g. TCA precipitation, in order to remove interfering substances.
Hi Par, my standard curve is always pretty good with r^2 values of 0.99X. I will give the spiking idea a go and see what happens. I have had a few suggestions to precipitate proteins now, so if I can't get it worked out, I will also be giving the precipitation a go.
Thanks for your suggestions!
Hi Joshua, I have been having your problem of no consistency between the Bradford assay and protein amount - did you figure out your problem? might help me figure out mine...
Hi Joshua! Let me know how you troubleshooted your problem because I will be doing similar experiments. Thanks.
Sutonuka Bhar, did you perfom some exosome protein quantification by microBCA assay? I'm planning to do some similar experiments.
Micro BCA is more sensitive than standard BCA, but the catch is that micro BCA is inhibited by the most common buffers such as RIPA or TBS, and many more chemicals. It is recommended a 1:10 dilution in ultrapure water before doing measurements with micro BCA. RIPA or TBS do not inhibit standard BCA at all, so I would go for standard BCA unless I do not have much exosome sample, which happens often.
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