My protein of interest was cloudy when it was purified in high concentration. But after overnight dialysis with storage buffer (20mM Tris, 150mM Kcl, 0.1mM DTT, 0.1mM EDTA, 50% glycerol), it becomes transparent with no sign of cloudiness. The protein was centrifuged at max rpm to check if there is any precipitation. There was no precipitation and activity of protein was really good. After a month of storage at -20dC, the cloudiness was seen again. What can be done to avoid such changes in protein.
(NOTE: The PI of protein is same as the pH of elution and storage buffer. Still, many literature including the crystal structure of protein, reported the same buffer conditions).
Probably, your protein concentration is close to the solubility limit, and the changes in condition upon dialysis were sufficient to redissolve the fraction that had formed a native precipitate. Since the pH corresponds to the pI of the protein, and the solubility of (native) proteins is lowest at their pI, slightly changing the pH is probably the easiest way to ensure that your protein remains in solution.
How the cloudy purified protein becomes transparent after dialysis against storage buffer?
First of all I would like to ask you if your protein was purified at homogeneity
Secondly
1) when you done dialysis of your protein solution in the buffer: (20mM Tris, 150mM Kcl, 0.1mM DTT, 0.1mM EDTA, 50% glycerol). We know that buffer with 50% glycerol is usually applied to concentrate protein in the dialysis tube solution. In fact, you will obtain a diminution of the volume of aqueous solution/water in the dialysis tube. Aqueous solution/water will be replaced by 50% glycerol. 2) In all, you, this dialysis will concentrate protein in 50%glycerol so your precipitate and the cloudy will be avoided by the protein solubilization in glycerol
In 5% glycerol your protein solution is not frozen; and your protein is not affected by 50% glycerol; since you showed that the protein activity is conserved after this treatment.
3) Yet, if your storage buffer has the same pH as the Protein pI. In that pH Isoelectric Point, the protein carries no net electrical charge and has the minimum solubility so the protein will precipitate again after storage.
In my view you should change the pH of the storage solution.
Your observations are very common. A change of buffer to conditions more suitable for your protein (i.e. your dialysis) can make the precipitate redissolve. Especially with charge-mediated aggregation. I don't know what you starting buffer is before dialysis but you might also see osmosis of water into the tubing, which will dilute your protein and also cause redissolving of the precipitate.
In terms of what can be done for storage - you're already using 50% glycerol - that's very high. Some proteins just don't take being frozen and defrosted very well - they don't have to withstand such conditions in the "real world". It might be that your protein falls into that category. Two things you need to make sure of if you want to try and improve the freeze-thaw behaviour of your protein:
1) Make sure to use liquid nitrogen for flash freezing. Proteins are much more stable when there is no vitrous ice formation and when they are frozen quickly. Then store at -80oC rather than -20oC.
2) Even though you say that buffer conditions for this protein have been published, it doesn't mean you can't make them better. And as other people have mentioned, having your pI equal the pH of the buffer sounds like asking for trouble. Unless of course the predicted pI is inaccurate. Optimise buffer conditions with an assay, if you have one set up, or with a turbidity assay, or a thermal shift assay.
Hi Pragna, I have a hunch that the 150 mM KCl is the problem.
K+ forms more insoluble salts than Na+
Prove me wrong and dialyze your protein into TBS with 150 mM NaCl and see what happens.
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