I’m currently using a protein (36 kDa) which needs to be unfolded during a labelling reaction. unfortunately the protein precipitates completely upon denaturation with EDTA which chelates the zinc ions holding its structure together. I’ve tried the reaction at 37, 40, and 55 degrees Celsius all with the same issue.
The exact same protocol (37 degrees, same edta:zinc ratio, time course) works for smaller constructs of the protein. I typically unfold, reduce, and then add the labelling reagent sequentially for 50 minutes each (total 2.5 hours shaking at 37). My protein concentrations have been between 20 and 200 micromolar, and the pH is maintained at 7.8 in 100 mM ammonium bicarbonate buffer (no salt) for optimal labelling.
I need the protein to remain in solution for downstream experiments after the labelling reaction. How can I denature the protein while keeping it soluble?
The protein pI is 7.06, which may be too close to the reaction buffer, especially considering that the other constructs had pi’s at 5.3 and 6.6. I’m considering testing a higher pH, unfolding with EDTA and a detergent, and increasing the salt concentration. Ideally I’d have as low a salt concentration as possible for downstream mass spec, and maintain the pH between 7.5 and 8.5 for optimal labelling with the different reagents. I’d appreciate any feedback or advice!
There are 2 main reasons why the protein could be precipitating: electrostatic interactions or hydrophobic interactions.
Increasing the salt concentration would probably overcome electrostatic interactions. You would have to remove the salt later, which is easy to do by passing the column through a desalting column or by dialysis. Changing the pH by several units could also help, but you have explained why this is a less desirable approach for you.
In my opinion, the more likely reason for the insolubility is hydrophobic interactions, since the interior parts of proteins are hydrophobic. You could probably keep the protein in solution using a detergent, but removing detergent later can be troublesome. There are a few detergents that are compatible with mass spec, however. The other approach would be to add a strong chaotrope, either urea or guanidine-HCl, at several molar. These are small molecules that can be removed later. You would have to experiment to find the minimum necessary concentration to keep the Zn2+-free protein in solution, especially if you don't want to completely unfold the protein, since refolding a completely denatured protein can be a challenge.
Please filter out the reason behind the precipitation first. pI, hydrophobicity, or inactivation of the metalloprotein part. Removing EDTA from denaturing solution, adding detergents or more preferably chaotropic agents into your buffer to promote solubility, or modifying the pH..All can be applied depending on the origin of the precipitation.
Eventually, you will indeed solubilize the reduced and probably alkylated denatured protein and 2nd consideration must be the labeling efficiency.
The interaction between your protein and payload should not be prevented or altered by the solubilizing agents you use.
Therefore choosing the optimal composition and concentration means finding the sweet spot during the labeling reaction is indispensable.
Removing all components may cause re-precipitation but keeping all intact in solution may prevent the payload from being bound. Thus, in addition to pH for labeling efficiency, potential non-specific affinities between your payload and solubilizing agents should also be taken into account.
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