I am working on a mesophilic protein (37 kDa and dimer in solution). I wanted to crystalize this protein and hence concentrated it to ~10 mg/mL. when kept on ice, the protein precipitated. kept the vial at room temperature for some time, the protein went back to solution. the buffer am using is tris HCl 20mM, pH 7. PI of the protein is 8. what might be the cause of this?
Dear lakshmeesha. I Think that if your protein due to the pi is so sensitive to ph shift around 7-8 you can replace tris with a different buffer ( hepes or phospate buffer are a good alternatives at ph=7) . Tris ph is sensitive to the temperature. ( https://international.neb.com/tools-and-resources/usage-guidelines/ph-vs-temperature-for-tris-buffer) And for proteins sensitive to ph variation is not the best storage buffer.
Good luck
Manuele
Do not understand your concern or what do you expect that will happen when you reduce protein solubility by temperature reduction. The opposite (solubilization) will happen if temperature is raised.
The pH of your Tris buffer increases substantially when it is cooled from room temperature to 0 C.
https://www.neb.com/tools-and-resources/usage-guidelines/ph-vs-temperature-for-tris-buffer
The pH at the lower temperature may be close to the pI, reducing the solubility of the protein. If you want to store the protein at pH 7, use HEPES instead of Tris.
Sounds like liquid-liquid phase separation. Ask coleagues or labs around that may know, or look for reviews all over the place. It could be telling something functionally important about your proten.
They appear as little droplets in a simple phase contrast microscope, in this case with capabilities of temperature control. They will go away if you increase the temperature. Since they are (very) concentration dependent, you may want to check the critical concentration. Do diulutions of the sample in the same buffer and measures simple scattering (ie 400 nm) in an absorbance spectrophotometer (of course at low temperatures). The scattering will dissapear with increasing temperature and with dilution.
If you only care about crystallization, forget about all this and keep screening. But means you are close to a crystal.
There are also proteins that are cold-sensitive and denature as the temperature drops too much (Wertheimer et al., 1944). That the protein redissolved at RT is quite typical for such proteins.
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