I purified the protein with Hi-trap column, the final buffer is 25mM HEPES, 500mM NaCl, 500mM imidazole. However, after overnight dialysis, the protein always precipitate. Anyone can help me to solve this problem ? the buffer for dialysis is 25mM HEPES, 500mM NaCl pH=8
Hard to know the exact cause of protein precipitation but, most of the time, it precipitates because the buffer is not adapted to this specific protein for its solubility.
Firstly, check if the protein do not precipitate in the Hi-Trap elution buffer after O/N incubation.
Secondly, things like temperature can enhance precipitation but I will consider that your experiments are made in the state of the art of biochemistry (low temperature, no air bubbles, gentle agitation...).
Finally, three things you might take care about for protein solubility is (1) ionic force (salt concentration) (2) pH and (3) protein concentration. I used to face this problem many time and the only thing I can suggest you is to try many different micro-dialysis and screen for different buffer chemicals at different pH and different salt conditions until you find a good one. If not possible, try to add additives that are known to enhance protein solubility such as arginine or glycerol. However, take care when using such additives, many of them are chaotropic compounds if used at high concentration. Those compounds might also be compatible with the measurements you're planning to do next.
You can also try to go back to the expression system you are using and fuse the protein to a solubility tag (thioredoxin, MBP).
good luck !
Antoine
Hi there,
It might also be a protein concentration issue. Diluted protein tends to precipitate easily.
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.
@Long Chen,
Check the pH of the protein prep and of the dialysis buffer. Are they different?
DEar Chyen
both buffers were at ph=8? Did you check the final ph of the buffer?
Which is the theoretical isoelectic point of your protein?
Do you know if the protein with the time tend to precipitate also in the elution with imidazole and therefore the precipitation is due to its intrinsic instability or if its really due to the buffer exchange.
In all cases i suggest you to speed up the buffer exchange using desalting instead dialysis.
in my blog: ProteoCool( http://proteocool.blogspot.com/ ) you can find some video that maybe can be usefull to you:
ProteoCool n°8 (Buffer Exchange Methods overview )
ProteoCool n°11(Rapid buffer Exchange by PD-10)
ProteoCool n°16: 3 Common mistakes in buffer preparation for protein purification
good luck
Manuele
Possibly, salt concentration in the dialysis buffer can be a salting out factor. Another significant reason could be too high concentration of your protein during dialysis. You can try diluting your protein prior to dialysis. Hope it helps.
Since the only difference between your elution buffer and the dialysis buffer is the His-concentration, I'd check the minimum His-concentration required to keep your protein in solution. Can the His be replaced by other small molecules? Typical osmolytes include polyols (sugars, glycerol) and amino acids.
Another question is whether a different pH or perhaps a lower salt concentration (say, 100 mM) could increase the solubility of your protein.
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