A number of methods had been developed for estimating protein concentration. Which method is most suitable in this case, and why?
There is not ONE perfect protein concentraion assay, it depends on the sample you have, interfering agents etc. For me, Bradford normally is enough, however, if you want to know the concentration of a specific protein (for example after purification) and compare it to another protein after purification, you have to consider that the number of aromatic or arginine residues has to be at least similar.
I find this summary very useful:
http://bitesizebio.com/10178/how-to-measure-protein-concentration-more-accurately/
Dewan Sumsuzzman, I agree with the previous answer. We don`t have ideal method without disadvantages, but for Western blotting and for sensitive enzymatic kinetics assays in our lab we often use Protein quantification assay by Roti-Nanoquant solution (https://www.carlroth.com/downloads/ba/en/K/BA_K880_EN.pdf) You can see a protocol here.
According to our experimental work, it is more sensitive that usual Bradford assay, and gives more precise protein concentrations. You can also check them by Nanodrop and compare.
I will advise you to try it.
Good luck with proteins :)
I would recommend this kit, easy to follow and reasonably priced.
https://www.thermofisher.com/order/catalog/product/23225
Can Kiessling, we also use BSA Pierce for all protein quantification assays.
So with Roth-Nanoquant solution it works perfect.
I agree with the comments given above. There is no a universal assay method that works perfectly for all proteins. When we select an assay method for proteins, we need to consider different factors, such as the physicochemical properties of proteins, components of protein solutions etc, that may affect the results of particular assay procedures.
Also, using the nanospec to determine protein absorbance is not ideal because the absorances are related to certain species of proteins i.e. mainly tryptophan and phenylalanine, while BCA/Bradford gives you more of a total protein estimation. Make sure you are within the linear ranges of that assay if you decide to use these methods.
May want to read
https://www.thermofisher.com/tr/en/home/industrial/spectroscopy-elemental-isotope-analysis/molecular-spectroscopy/ultraviolet-visible-visible-spectrophotometry-uv-vis-vis/uv-vis-vis-instruments/nanodrop-microvolume-spectrophotometers/nanodrop-protein-quantification.html
and
https://tools.thermofisher.com/content/sfs/brochures/TN52643-E-1214M-NanoDropProteins.pdf
and
https://www.researchgate.net/post/Should_we_use_nanodrop_for_total_protein_amount_measurements
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