Dear all,
I am purifying a His tagged protein (theoretical pI 6.06). The elution buffer contains 50 mM NaH2PO4 pH 8, 300 mM NaCl and 300 mM Imidazole. I have to perform CD analysis of the same protein and hence I perform dialysis in the buffer containing 50 mM NaH2PO4 pH 8 and 50 mM NaF. I observe a cloudy solution after 12 hours of dialysis. How to get rid of this? Any suggestions.
Dear Swapnil,
Please check if your protein has cysteine in a sequence, It could be oxidation of cysteine bridges, probably you will need add reductants (DTT, mercaptoethanol or TCEP).
And in general you can use desalting column for buffer change - it will be much quicker than using dialysis, I personally prefer desalting column rather than dialysis, for my proteins I also got several times precipitates during dialysis and no problem with desalting.
As for desalting column - nowadays a lot of vendors offers this type of columns - PD10 (GE Healthcare), Econo-Pac 10DG (Biorad) and others. Or you can prepare home made column using Sephadex G25.
Yes, As suggested earlier by Rafil, it is better to desalt the solution after Nickel column using a PD-10 column and if concentration of the sample is an issue, better to run it through a home made Bio-rad P6 column with 90-95% recovery. In the process, the protein can be collected in the buffer of choice. This would be a faster and better method than dialysis.
The concentrated protein may require high salt to remain in solution if it tends to aggregate due to ionic interactions. A more dilute protein solution may require less salt. The pH may have an effect on this phenomenon, as well. Adding salt back to the cloudy solution may restore it to clarity, if this is the explanation. It doesn't have to be NaCl. Choose a salt that is compatible with far-UV CD spectroscopy.
Another possible explanation could be the temperature. Some proteins, during refolding, tend to aggregate if it happens too fast. Low temperatures slow down that process and sometimes prevent aggregation. Try dialysing into a cold room with temp 4-8 oC.
The stability of proteins, and their propensity to precipitate, varies widely in different buffer compositions. The protein's concentration can also influence solubility. My initial suggestion is to try the same buffer background, but with additional L-arginine at say, 50mM conc. Otherwise you might have to screen a wider range of buffer compositions to promote your protein's solubility.
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