I purified my proteins from plants in 7M urea buffer through nickel purification. Then I changed purified proteins into a native condition buffer for overnight (Tris and phosphate) for other assays. Should I worry about degradation once proteins are purified and do I need to add protease inhibitors in this native condition buffer ?
If you purify it then you should not have any proteases along. Nonetheless all proteins in solution will tend to aggregate due to the hydrophobic effect, thus to slow this process keep your solution cold or remove water completely by freeze-drying.
Proteolysis can be a problem in some cases if the proteases carry through the purification and if they regain activity during renaturation. If it occurs, add the protease inhibitors to the protein while it is still in urea.
Thank you for both your answers. I would like to know how likely proteases tend to purified together with the target proteins through affinity purification??
If proteases have as many surface histidine residues as your desired protein then they will co-purify. If this is not the case then an imidazole gradient should be able to separate them.
You don't have to worry about it but go ahead and test it. Only if you check whether YOUR protein is protease sensitive after the purification or not, you will know.
Otherwise you will have to rely on speculative or in some cases wrong guidelines. Every protein is different, some are very sensitive (especially after refolding) some more resistant, but nobody can tell you the answer for your protein.
The statement above is not exactly correct. If you overload your SDS-PAGE, you will realize how much other proteins are still there, especially after one Ni-column, let alone the nucleic acid where you also have plenty of DNA/RNA-binding proteins. So it is very likely that there is always some protease activity (dependent on purity, harvesting time etc.).
Remember that not only proteases but also incomplete translation products can lead to fragmentation, so if protease inhibitors show no benefit, this could be one reason.
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