The problem is solved differently for different proteins, depending on their physicochemical properties.

The major danger of abrupt changes in urea (or any other denaturing agent you may use) is protein aggregation. As a general case, one expects protein globules to form spontaneously as soon as the denaturant is removed. The trick is that often a protein needs more time and a certain path to acquire the native fold, whereas misfolded, inactive species are produced rapidly in a variety of possible folding paths. When you decrease urea stepwise you produce a kind of path for proper folding, giving the protein both time and the opportunity to go back to correct the fold. When you drop urea sharply aggregates are formed quickly and, most tragedically, irreversibly.

Remark that this situation is not universal! Some proteins (among them rhodanese) are most susceptible to aggregation, for example, at 4M urea. The best way to renature a protein is to be found empirically.

Hello,

beside the fact mentionned above, you should think of why you are using 8 M urea. Urea is a chaotropic agent that will defolded proteins. Although 2M is still high you might increase the chance of protein misfolding and thereby precipitation. Thus best is to get rid of Urea in a linear fashion in order to permit the best refolding of the protein.

Best

The problem is solved differently for different proteins, depending on their physicochemical properties.

The major danger of abrupt changes in urea (or any other denaturing agent you may use) is protein aggregation. As a general case, one expects protein globules to form spontaneously as soon as the denaturant is removed. The trick is that often a protein needs more time and a certain path to acquire the native fold, whereas misfolded, inactive species are produced rapidly in a variety of possible folding paths. When you decrease urea stepwise you produce a kind of path for proper folding, giving the protein both time and the opportunity to go back to correct the fold. When you drop urea sharply aggregates are formed quickly and, most tragedically, irreversibly.

Remark that this situation is not universal! Some proteins (among them rhodanese) are most susceptible to aggregation, for example, at 4M urea. The best way to renature a protein is to be found empirically.

you can also dialyse your solution against decresing urea and be carefull, if you observed a slight precipitate, stop dialysing against urea and dialyse against your buffer with 0,1%SDS...this way you can decrease urea. I am using this to get rid as much as possible of urea to allow coupling of insoluble protein to CNBR sepharose beads.

What if my protein does not aggregate if I reduce the Urea rapidly by dialysis? Is there a chance of mis-folding?

refolding of a protein using dialysis is always a challenging task..and assesing the fraction of refolded protein is also of concern. from my experience, it is better to try to optimise the expression of the protein within a given host (vbaculo, ecoli and other) than trying  ti refold a proteine.

I`m trying to do refolding by dialysis after solubilization with 8 M urea. I do dialysis with 6, 4, 3 and 2 M urea, but proteitn precipate at 3 M urea buffer.

Any suggestion on how to prevent precipatation, how long should I do dialysis in each step?

Hi guys, I'm trying to use dialysis to get rid of Urea 8 M. I have aggregation and protein precipitation on the membrane. I would like to try to perform dialysis  a buffer containing salt (TrisHCl or ammonium bicarbonate). I have to use MS-compatible buffer since I am going to do mass spec after! Can somebody confirm if this is the right way to do it to avoid as much as possible protein precipitation and aggregation?

i suggest to dialyse step by step against 6 then 4 then 2 then 1Murea  plus added 0,5M NaCl..and see if this is suitable for your mass spec...if the sample is for mass spec after tryspsin digestion, why not simply run some of your protein in gel?

Hi Valeria,

Do you digest your protein with trypsin before your mass spec?

Alain

In the dialysis of proteins is very useful to perform a steps-process in order to give the protein enough time to refold properly, this is a slow procedure, I would recommend 3-4 h at least in every step. It is important also to diminish the protein concentration, because if protein is prone to aggregation, it will do if it is not diluted enough. Steps and low concentration is essential. Each protein is different, therefore the protein concentration at which it is possible to carry out the refolding can change for each one.