What is the maximum concentration of Urea that I can add without denature the protein or what is the minimum concentration of urea that is needed for a protein to be denature?
We have always used an 8M concentration but we have seen that 6M is also used. I would like to know how much is the minimum that can be used to achieve protein denaturation
There is no way to give a general answer to your question; the concentration of chaotropic agent required to denature a protein is completely dependent on the protein. Some thermophilic protein are not denatured even by saturating concentration of urea, nor by SDS; some others may be denatured with 5 M urea. Practically, the concentration of urea is limited by its solubility to about 8 M. More powerful denaturants are guanidine hydrochloride or guanidine isothiocyanate. They are however more expensive than urea and furthermore, their high ionic strength precludes their usage for SDS-PAGE analysis
I believe that the 8M urea standard is based on convention and convenience rather than science. The ratio (w/w) for urea to water is ~1:1 (60 g/mol * L) for 8M urea, just a shortcut that's easy to remember, is most definitely overkill, and I don't know if you can dissolve any more than that without additives or supersaturating the solution.
Dear MAria Teresa
as Pierre already told you, there is no a universal answer to your question because it depends from the intrinsic stability of each protein. For example some enzymes, as Cu,Zn SOd1 that contain S-S bonds that stabilize its folding it is active in 6M URea while the most of proteins are unfolded in this condictions.
If you have the purified protein you can check it, by incubating the protein in different urea concentration and:
- check the tm shift using DSF ( Article Real-time protein unfolding: A method for determining the ki...
)- If you protein contain exposed aromatic residues as W or Y you can also check the change of fluorescence emission in presence of increasing concentration of urea
eg Fig 3 on Article Human Sco1 functional studies and pathological implications ...
or http://www.jbc.org/content/274/47/33251.full.html
- Of course if your protein is an enzyme and you have in your hand a methods to monitor its activity, you can also perform it in presence of increasing concentration of urea to see which is the minimun activity that inhibit it.
In case you do not have the pure protein, but you would like to understand which is the minimal urea concentration to extract it from IB, you need to perform serial extraction with incresing UREa /1M, 2M, 3M, 4M, 6M, 8M) and see which is the best. In same rare cases, 8M urea is not enough and you have to use Gn-Hcl and reducing agents.
Gn-HCl work well, but you cannot load the sample direclty in SDS-page. You need to dilute it in a buffer with urea to reach a final Gn-Hcl conc
Depends on type of protein. Structural proteins tend to tolerate higher concentrations of urea while enzymes can be denatured using even 3M urea solutions. If you have purified protein, I would recommend incubate protein in increasing concentrations of urea from 1M to 5M for 1hr, 3hr and 12hr. Record the CD spectra and compare it with spectra of protein without urea. It will give you clear idea about minimum urea concentration required.
The effect of the chaotropic agents on the protein is, as stated by others, dependent on the characteristics of the protein, but also on the kind of chaotropic agent used.
The two most widely used ones are urea and guanidine hydrochloride. Urea acts more on the hydrophobic and non-polar regions of a protein, while guanidine HCl acts more on the polar and charged regions.
I worked once on a viral protein that needed a combination of high concentrations of both urea and guanidine HCl to be denatured properly. The biggest problem was preparing the solution, as the volume of powdered reagents to be dissolved was larger than the final volume of the solution.
Hi Maria,
Urea and guanidine HCl are both medium-strength denaturing agents and are easily removed by dialysis and ultrafiltration. They have a strong reversible denaturation effect on hydrogen bonds of inclusion bodies. I usually use 8M urea or 6M guanidine HCl. The actual situation should be based on properties of the protein, but usually 6M guanidine HCl is sufficient to denature the protein.
Hope it helps!
hello Maria,
Can you please share your experience here. I need to know what will be the best and optimum volume of 4M urea to denature a peptide/MHC I complex.
Thank you
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