6-8M urea should enough to denature your protein. For long-term storage before refolding studies, I would use 6M Guanidine HCl. If you prefer urea, I would freeze the samples at -80 to avoid chemical changes.

Do remember that urea is a chaotrope and at moderate to high concentrations denatures proteins/protein domains. Its chaotropic nature however does actually arise from its tendency to increase total entropy, ergo, it can be countered by storage at very low temperatures and as such shouldn't denature proteins as efficiently than at ambient temperature. Just remember that at low temperatures you may cause the urea to precipitate if you're using saturated solutions. To prevent the denaturation of your protein you may add agents that promote protein stability.

You can qualitatively check the presence of your protein secondary structures by CD spectroscopy. Also, you may want to run a thermal denaturation assay to see if your denaturation midpoint changes in the presence and absence of urea upon prolonged storage. Consider doing these assays with differently stored batches of proteins (+4 C, -70 C etc.). Even better if your protein is an enzyme, as you can check enzymatic activity to elucidate if the quantity of active protein decreases over time. Also, with simple spectroscopic measurement at A280 you can crudely check if your protein is unfolding, as the absorbance at A280 changes when the proportion of folded and unfolded protein changes at constant concentration. ProtParam etc gives you the absorbance coefficient for an unfolded protein, whereas for folded proteins the coefficient is slightly different and has to be empirically determined.

As for storage time - to my understanding proteins can be stored at -70 for extensive times. At +4 C some proteins tend to lose their activity/folding or may even degrade due to autolysis. Then again, some proteins do not. How a protein behaves upon storage is really protein-specific. If your protein withstands freezing, it would be the best way to store your protein for longer times. I usually snap freeze with liquid nitrogen (to minimize water dynamics that may affect protein structure) and store at -70 C.

OK, thank you for your answer.

Just to make sure. I use 8M urea solution for solubilizing my protein, and I know that it is unfolded, denaturated, thats fine. I do not need folded protein for now, and If I will need I can try to refold it again.

To my understanding 8 M urea should keep your protein (and others) quite strictly denatured. Refolding should not change whether you denature your protein with urea or some other chaotrope. However, I have read that sometimes unfolded proteins undergo autodegradation, which can be prevented (or at least decreased) using additives. Depends a little. For instance in SDS unfolded proteins are quite stable for some time even at room temperature. I think that it really depends on *how* you store your protein.

ok, but leave folding and unfolding for now. What I meant from the beginning is structure (chemical structure) changes, like I heard that urea can change amino groups into different group. I just wanted to proof it. If it safe to keep my protein in 8M urea in -20 or -80 for longer time (like 2 months).

I hope everyone know that urea buffers have to be prepared freshly because of isocyanic acid which is forming in urea solutions.

And if there is any assay to test if there is everything ok with my amino groups in protein.

I don't know about specific assays, but qualitatively you should be able to probe chemical differences in your protein using mass spectrometry (ESI-TOF). Just remember to do it in desalted and reducing conditions to reduce ion adducts and oxidated species. This is something I routinely do for my proteins.

Beware carbamylation

Marcin

Urea solutions slowly decompose to form cyanate and ammonium ions. The cyanate ions can react with amino groups from proteins (Prakash Loucheux Scheufele Gorbunoff and Timasheff Arch Biochem Biophys 210 (2) 455-464). You should use a fresh recrystalized urea solution. Some people add 5 mM Glicine in order to ”compete” with protein and reduce sample modifications.

Lucrecia, Is it also happens when my samples in urea are in -20 degree?

And for how long I can store for example buffers for chromatography with 6M urea, 1 day in +4 is fine?

I have found some informations here http://www.ionsource.com/Card/carbam/mono0005.htm

i do not know how reliable it is. they are suggesting acidifying the soltution with 100mM HCl but in my case I have to keep my pH above 8.

Marcin, heat has a lot to do with cyanate formation and carbamylation. I think that its formation will be reduced at low temperatures. Add glicine to protect your protein and check its molecular weight by Mass spectrometry after storage.

Beware of pH at -20°, some buffers change a lot their pH!

Marcin... It is much better if you make a fresh solution everyday. Hard work (but higher-quality results!)

Yes with buffers there is no problem, I can make them everyday fresh. The point is that I need many different samples in urea, purified with chromatography at once for experiment. So I do not know If I should store them in Urea after chromatography or for example precipitate in ethanol and store like this and after all purifications just dissolve it in urea. Maybe it will be better, and I won't loose a lot of protein I think during it.

ohh and I know mass spec will be excelent for this but I do not have access to it for everyday purpose... I guess I won't see any difference in mass on a gel right? It is too small...

and one think about carbamylation. If lets say my Lys is carbamyleted, so this carbamoyl group can be carbamyleted once again? I mean there is still amino group so it can produce some kind of chain?

I don't think it forms a chain. You can think about it in a way that otherwise in a solution of urea it would react with itself to form a polymer as time passes. This is possible, but requires a high concentration of isocyanic acid (in other words, a lot of time). The carbamyl group formed at the end of a lysine side chain to my understanding is not (as) sensitive to further carbamylation as a free amine group would be.

I agree with Arne about carbamylation.

Quality control by CD will be a good choice if modification of Lys residues leads to a structural perturbation.

Correct me if I'm wrong here (it's been a while since my last chemistry lecture) but in a carbamylation reaction the amine group of the lysine acts as a nucleophile and attacks the electrophilic carbon of isocyanic acid (or isocyanate at near-neutral pH) ? Logically speaking this could be prevented by acidifying the pH, as the Lys R-NH2 equilibrium tips more towards R-NH3+ and prevents the free electron pair from doing a nucleophilic attack.

To the best of my knowledge, proteins usually suffer from denaturation in the presence of urea, there is a limit of concentration that will unfold totally the protein that will depend on the type of protein. Be aware that sometimes the process is irreversible, like in thermal denaturation.

If you have access to a CD (circular dichroism) equipment, you will be able to follow the unfolding process (via the secondary structure of your protein), and this will give you an idea about the structural stability or stability under your storage conditions. If your protein is an enzyme, I would advise you to run some enzymatic experiments in order to check stability, but I assume that might not be the case as that is the straightforward answer.

Calorimetry would be another option for checking stability, but this may destroy your sample in the analysis process via thermal treatment and might not be an option if you have little amounts of protein.

The question... Urea Causing storing proteins in any changes in structure? is very relatives, due to significant structural changes in proteins as a result of the urea concentration dependent chemical agent used. At higher concentrations of 4.0 M (25 ° C) significant changes begin to noted by Circular Dichroism (CD) in some regions of the protein. However, at low urea concentrations (≤ 400 mM) the initials steps of the molecular mechanism of Urea–protein interaction passes through the establishment of a three center four electron adduct with polar or charged residues affecting enzyme activity but not the apparent three-dimensional structure by CD (Almarza et al., 2009; Almarza et al., 2013)

Quite agree with you Jorge. But sometimes lower concentrations of urea, like 3 M (at 25 ºC) affect the secondary structure of the protein.

Marcin did not say what concentration he had in his sample.

Another point he wanted to clarify was storage conditions. That depends highly on the intended use of the protein. If you require native conformation, for instance for an enzyme, or if the protein is to be further analyzed for its constituents (peptides, structure...) or any other analytical procedure. There are cryoprotectants that can be used. Or if you store it at 4 ºC and you want to preserve activity, sodium azide can be added in order to avoid microbial growth and potential degradation, or peptidase cocktails to prevent enzymatic digestion of your protein. It is usual to combine both.

For a long period of time, I would lyophilize the protein and store it at -20 ºC if possible.

Hey, thanks for replies. I am using 6M and 8M concentraions (I think I mentioned it somewhere here). I need later those proteins for refolding experiments (stop flow exp).

Why damage your protein by storing it in urea?

6-8M urea should enough to denature your protein. For long-term storage before refolding studies, I would use 6M Guanidine HCl. If you prefer urea, I would freeze the samples at -80 to avoid chemical changes.

Storing protein in Urea chemically modifies the Lysine residues because of the isocyanate that occurs through urea degradation.