Recently, I purified DnaA proteins from two different species via nickel-affinity chromatography. Based on their respective theoretical pI values, I prepared tailored purification buffers. However, precipitation occurred in two elution tubes during protein elution. Could this be caused by excessively high protein concentration? If precipitation is indeed due to high protein concentration, does this indicate that ultrafiltration is unsuitable for removing imidazole from the protein solution? Alternatively, should I ensure that the protein concentration during buffer exchange never exceeds the concentration observed in the E2 and E3 tubes (as shown in the figure)?
You can run SDS gel on the precipitates to make sure that it is your protein that's precipitating. If that's the case maybe dilute the elution fractions as soon as they are eluted. Then I would remove imidazole by dialysis o/n, unless you have another purification step following Ni. It looks like you may need it.
It may be possible to increase the solubility of the protein by changing the conditions, such as changing the pH or the salt concentration, or by adding a detergent. However, the easiest approach would be to lower the protein concentration.
It is sometimes observed that a protein precipitates during dialysis to remove the imidazole. This does not necessarily mean that the protein can't be used. It may dissolve again when diluted. It's at least worth checking. In any case, changing the dialysis buffer conditions may be beneficial to improve the protein's solubility.
As Adam B Shapiro said try changing salt concentration. I have found that a little EDTA can sometimes help preventing aggregation after IMAC. Presumably it is caused by leaking Ni. You will have to add it in the fractions - not in the running buffer. Are you doing step elution? Gradient elution may help keep the concentration down if that is the problem.
Hello professors, thank you for your suggestions. Based on my previous experimental results, I confirmed that the precipitate is indeed my target protein. I attempted to determine the optimal wash concentration through gradient elution. After nickel column elution of the protein, precipitation was observed in the second and third elution tubes but not in others. Today, I combined the protein solutions from the remaining elution tubes and concentrated them using ultrafiltration tubes. I noticed that even after concentrating to a very small volume (e.g., from 7 ml to 1 ml), the solution remained clear without turbidity. This suggests that the precipitation may not be caused by high protein concentration? Regarding the protein precipitation, my senior lab member suspects that the DnaA protein may have bound a significant amount of nucleic acids. Is this suspicion reasonable? If this is possible, could I remove some nucleic acids during nickel column purification by using a high-salt buffer?However, I observed that the solution became turbid when I attempted to remove imidazole. My protein was eluted in a buffer containing 50mM HEPES-KOH pH 7.5 (25°C), 500mM NaCl, 500mM imidazole, and 10% glycerol. When I used an exchange buffer of 50mM Tris-HCl pH 7.5 (25°C), 200mM potassium glutamate, and 10% glycerol, the protein solution turned turbid immediately upon adding this buffer. When I used an exchange buffer of 50mM HEPES-KOH pH 7.5 (25°C), 500mM NaCl, and 10% glycerol, the solution remained clear initially after adding the buffer but became turbid after a few minutes. Re-adding the elution buffer containing imidazole failed to restore clarity. What could be the reason for this? In this situation, can I use a dialysis bag to remove the imidazole?@Asli Ertekin@Adam B Shapiro@Bo Pontoppidan
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