Elution buffer composition is- 50mM NaH2Po4, 300mM NaCl, 250mM immidazole at pH 8.
PI of my protein is 6.7. I purified My protein using Ni-NTA resin and run sds page and till the completion of gel I kept my samples at 4*c. When my gel got over I checked and confirmed the elution bands at desired size, I went to take my samples back from 4*c and planned to kept at -80*c for further use. I found precipitation in the elution fraction number 3 and 4 (Having more protein, as per sds page result). What should I do to avoid it?
The protein concentration in the most concentrated fractions may be higher than the protein's solubility under those conditions. It may be sufficient just to dilute the protein. Changing the buffer conditions may also help, such as lowering or raising the salt concentration or changing the pH.
If Adam B Shapiro's suggestions should not work, try replacing the phosphate with tris base or a sulfonic acid (e.g., HEPES, etc.). Also, including a chelator like EDTA at 10mM or so might be worth a try.
Adam Shapiro is spot on - it is likely the high concentration which is causing precipitation. One way to lower it is eluting with a gradient or at a slightly lower imidazole concentration. We have also seen that some proteins don't seem to like imidazole - they must be transferred to another buffer directly after elution.
Another thought I had is that the pH may be too high for the stability of the protein if you did not adjust the buffer pH after preparing it, since imidazole is basic.
I think the temperature at which you purified the proptein is what is affecting it. Did you know to purify it in a cold room? Another factor is the pH
Adam's answer sounds really convincing, but I just want to ask if your protein is a membrane protein by any chance? This would change a lot of things.
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