What could be the cause of immediate formation of cloudiness upon addition of Zinc ions in a purified His-tagged metalloprotein? Surprisingly, the cloudiness which is more like protein aggregation disappears after a few minutes.
50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 250 mM Imidazole, which constitute the elution buffer.
The protein in the elution buffer was then spiked with 5 mM ZnSO4 and 1 mM TCEP.
Why did you add ZnSO4? Did you try to remove contaminating nucleic acids?
Sulphate salts, such as ammonium sulphate, sodium sulphate and zinc sulphate, are important protein precipitating agents. They often cause precipitation of proteins via selective salting-out effect. They are used to purify proteins. ZnSo4 is also used for selective precipitation of nucleic acids depending on its concentration.
Addition of ZnSo4 to your purified protein might cause precipitation of the protein. You can collect the protein pellet by centrifugation and re-suspend using a buffer that maintain your protein in its stable and soluble form.
The concentration of the salt is very important. You need to use an optimum salt concentration to achieve your goal. High salt concentration will cause protein aggregation and low salt concentration are not able to precipitate the protein, thus target proteins will remain in solution, resulting low protein yield following precipitation and re-suspension.
I think 5 mM ZnSO4 is too low to 'salt-out' proteins.
My guess is, since the pH of the buffer was basic, Zn ions formed insoluble zinc hydroxide.
More needs to be probed.
Might be because addition of Zn altered the oligomeric state of the protein. There are many studies which talk about metal dependent oligomerisation of proteins.
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