Hi All,
Recently, I purified 8 proteins and stored at -20 with the elution buffer containing 20 mM Tris, 250 mM NaCl and 200 mM Imidazole. I autoclaved the buffer, so I do not know whether the buffer is still fine or not. I did not add triton X and glycerol. My protein concentration was around 1 to5 mg/ml. I stored my proteins at -20 and next day I took them out to thaw. Unfortunately, All my proteins formed aggregate. I could see the white clump. So, I span down and re quantify. protein concentration was reduced by 90%. Could you please share your opinion on how to prevent aggregation from freeze-thaw? If my protein is stable at 4C for 10-15 days, should I still need to store it at -20 or -80C? I am planning to elute my protein in Sodium phosphate buffer with 0.1% Triton-X and 5% glycerol. Should I also use NP40, PEG6000 along with Triton X.
Hello Abir, there is no general rule, some proteins like salt some other don't, so you have to try both. Make sure also that the isoelectric point of your protein is not close to the pH you use, and if it is, change the pH of your buffer to at least 1 pH unit away from it, and be careful that the pH of a Tris buffer is quite temperature dependent. Alternatively, imidazole can promote aggregation in some instances, so you can try to keep the salt but remove the imidazole (sometimes diluting the sample with elution buffer without imidazole just after it exits the column to something like 50 mM imidazole does the trick). Indeed it is possible that aggregation begins even before freezing and that freezing-thawing just makes it worse. Finally, try to flash-freeze as much as possible (in liquid nitrogen or ethanol+dry ice ), slow freezing can damage proteins.
Olivier
It depends on the protein, but most likely it's the freezing with high salt (including imidazole) that's killing it. Cryoprotectant does not necessarily help with that. I think you should do a buffer exchange into a buffer that matches the environment where your protein is normally found, and then add cryoprotectant too. It is most convenient to do this on the same day with a G25 desalting step into the new buffer. You could also use overnight dialysis, or simple dilution.
John Schloendorn Thank you very much for your opinion. I was reading a protocol from Qiagen and it says that a high concentration of NaCl helps to prevent aggregation. I will do buffer exchange and add cryoprotectant as well.
Firstly, if your protein is stable at 4°C, then there is no need to freeze it. Secondly, dialyze the protein at 4°C to remove imidazole before storing it. If you intend to use sodium phosphate buffer, then use 50mM sodium phosphate buffer, 250mM NaCl & 5% glycerol in your dialysis buffer. Glycerol helps to stabilize proteins.
High salt can help when keeping it in solution. When freezing, it's deadly!
Hello Abir, there is no general rule, some proteins like salt some other don't, so you have to try both. Make sure also that the isoelectric point of your protein is not close to the pH you use, and if it is, change the pH of your buffer to at least 1 pH unit away from it, and be careful that the pH of a Tris buffer is quite temperature dependent. Alternatively, imidazole can promote aggregation in some instances, so you can try to keep the salt but remove the imidazole (sometimes diluting the sample with elution buffer without imidazole just after it exits the column to something like 50 mM imidazole does the trick). Indeed it is possible that aggregation begins even before freezing and that freezing-thawing just makes it worse. Finally, try to flash-freeze as much as possible (in liquid nitrogen or ethanol+dry ice ), slow freezing can damage proteins.
Olivier
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