My His-tagged purified protein contains 50mM sodium phosphate, 300mM NaCl, and 8M urea at which the protein was eluted at pH 4.9 and pH 4.3. What should be the ideal dialysis buffer composition to remove 8M urea?
I am using 50mM sodium phosphate, 300mM NaCl, and 8M urea at pH 4.9 and pH 4.3
Does NaCl affect protein function? Do I need to remove it too?
Because the protein was purified in a denatured state in 8 M urea, it has to be refolded. If you try to remove the urea by a single step of dialysis, the protein will probably precipitate because it will not be able to fold correctly during the short time that the urea disappears.
Refolding a protein by urea removal using dialysis is normally done in several steps of decreasing urea concentration. At the same time, the buffer should contain a substance that helps the protein refold. A common one is 0.5 M arginine. The protein concentration should be as low as feasible to minimize aggregation. Nevertheless, at the end of the process you will almost certainly have to perform gel filtration chromatography to remove aggregated protein.
NaCl is often included to help prevent protein aggregation through charge interactions. Typically, the concentration is 150-300 mM, but it may not be necessary during the initial stages of refolding.
You will need a pH buffer. The buffer shoild be chosen to maintain the pH at the value that is where the protein is most stable. This is usually around neutral. Tris or HEPES or phosphate at pH ~7.4 are suitable for cytoplasmic or blood-borne proteins. If your protein is normally found in an acidic environment, then an acidic buffer is probably preferable.
If your protein normally contains disulfide binds but is in a reduced state after purification, you will need to include a redox buffer, such as 3 mM reduced glutathione + 1 mM oxidized glutathione.
Because large volumes of buffers are need for dialysis, which can get expensive, you could consider other methods of refolding that use less. These include rapid dilution and on-column refolding.
I suggest you read some literature on protein refolding methods before you set out on this journey.
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