I purified my protein by nickle column with FPLC. My base purification buffer had Tris 50 mM, NaCl 500 mM, Imidazole 40mM, urea 8M, 2Me 5mM at PH 8. I removed urea by gradient and eluted protein by imidazole 500mM, Tris 50 mM, NaCl 500 mM, 2Me 5mM. the purification was good. When I desalting protein by dialysis, the protein get aggregated. I changed dialysis buffer from PBS to Tris 50 mM, NaCl 150 mM at PH 8 and the time of dialysis was 4 hour, but I saw turbidity.
Mohsen Soltanshahi , when i have he same problem, i add 0,1-0,2 % sarkosyl sodium (or SDS) to protein before dialysis and in dialysis buffer too.
You go from 8M urea to 0M urea in one step. This is too fast for most proteins.
Optimal is to change in serial dialysis steps of 2M downwards; 8, 6, 4, 2, to 0M.
You could also try just adding a single step with 4M urea - sometimes this is sufficient.
I would also try to add 1 - 5mM EDTA to your first and second step in the dialysis, to get rid of metal ions.
A few observations, that I'm pretty sure you have thought but it's better to be remember:
* Since your protein is purified in denaturing conditions, you must be sure that the protein is well folded after purification, sometimes the observed aggregation is due to some protein population is not well folded or is kinetically unstable. Just keep in mind.
* Stay away from the pI from your proteins (I mean, is the pH 8.0 the correct for your protein?).
* For many proteins is needed reduction conditions (mainly for brake down disulfide bonds in aggregated form).
* If glycerol is added, you need increase the time of dyalysis.
* Arginine in high concentration is sometimes useful to prevent aggregation (although for next experiments could be a problem -spectroscopy, activity, etc...-).
* Exist many possibilities determining which are the appropriate conditions for your protein, it can take days, but if your protein is the "protein of your dreams", it's worth trying (you can check this paper: Anal Biochem. 2003 May 15;316(2):223-31). To assay different conditions, microdialysis on a battery of different buffers is a good option.
* Have you checked the protein concentration before dialysis (I know you have imidazole and it is not exact, but at least one approximate)? You must differentiate if the aggregation is due to a high concentration of protein or just because you don't have the correct stability conditions for it (I mean, buffer, pH, additives, temperature ...)
Good luck.
Thank you so much. I have another question. I want to produce a monoclonal antibody. So I should inject to mice and also do Elisa by this protein. I know that my protein will aggregate at -20. My question is, after dialysis, what is the best compound for storing the protein at -20? Can I add glycerol 50% or 20%, sucrose...
I want to use a compound which not interfere with other steps.
If you sterile-filter it, you can store it at RT for a while.
Other wise, =< -65C. For proteins, in general, -20C is usually one of the worst choices, as this is too close to the glass-transition point for many proteins.
Mohsen Soltanshahi , and don't forget about freezing point of aqueous solutions
https://en.wikipedia.org/wiki/Glycerol_(data_page)#Freezing_point_of_aqueous_solutions
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