Hi everyone,
I am trying to find a cheap way to compare the proteomic composition and abundance of some bone samples. In particular, I am expecting to see a reduction in the abundance of some of these proteins as a result of different treatments that I will perform on these bones.
Since I have to exclude HPLC/MS-MS (too expensive) and any other mass spectrometry technique, and since I believe that an SDS page will results in a complex smear (more than 50-60 proteins in the mixture) and a 2D-GE will be too complicated to find differences between my samples, I was thinking to simply quantify my samples in order to compare them. But, since I am expecting some proteins within this mixture being more denatured in same samples than in others, my question is: are protein assays (e.g., Bradford, BCA, etc.) able to provide different quantification results for denatured vs folded proteins? I don't know exactly the chemistry behind them, so anyone able to help me will be extremely appreciated! Any other suggestion will be appreciated as well!
Many thanks!
Hi there,
The protein assays you intend to use are all denaturing so basically they won't make any difference between folded and unfolded proteins. Denaturation is generally making protein less soluble so the easiest would be to centrifuge samples prior assay in order to eliminate precipitating material.
Dear Sir. Concerning your issue about the evaluation of protein denaturation with protein assays. I think the following below links may help you in your analysis:
https://pdfs.semanticscholar.org/a0b2/d6c375092dedd05366e8c2774d34b03bc7a8.pdf
https://www.thermofisher.com/sa/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-protein-assays.html
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4631703/pdf/nihms-730075.pdf
Thanks
Thanks for all your answers! Extremely appreciated! After reading all what you suggested, I am thinking to actually combine two methods to try to find differences.
One will be using an assay to quantify proteins (I'm expecting some proteins being denatured and some other proteins just missing because of their leaching out from the bones during the treatments), so my question is: which one will you suggest? BCA? Bradford? I am just worried that they will interact not only with whole proteins, but also with peptides originated from protein fragmentation due to their decay, so maybe this won't work for my aims.. I'll centrifuge the sample as suggested by Dominique Liger (any suggestion about speed and time?) to try to get rid of the denatured ones.
Then, the second approach could be using the fluorescence spectroscopy, as I found also in the thesis that Isam Eldin Hussein Elgailani suggested.
What do you think? Many many thanks again!!
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