I totally agree with Ranjan! Doublecheck your pI and try lower and higher salt. Additives like glycerol have also been described in the literature to reduce aggregation and to stabilize certain proteins. Why not giving 5-20% glycerol a go? In addition, you can try to do a wash / on-column dialysis step (if you have some sort of affinity tag to bind the protein to a column) and run a urea gradient from 1 to 0 M over lets say 10-20 cv before eluting in your dialysis buffer. I am not an expert on protein refolding however I think many people also add arginine to assist folding. Also temperature is a factor that probably needs to be considered ... did you do your dialysis in the fridge?

I also try to avoid phosphate buffers like PBS since phosphate has a tendency to from salt precipitates. Did you check if your precipitate is your protein or just salt?

Another thought would be construct design or expression cell line.... since you have your protein in 1 M urea it may not be properly folded? Was it ever soluble without urea? Did you try different expression cell lines including extra plasmids for rare t-RNA codons?

If nothing helps the hard truth is often it is actually better to redesign the epression constructs in order to obtain a soluble protein.

Best of luck!

Is the pH of both buffers same? if not how different they are..as during dialysis if it passes through the protein pI then it will surely precipitate. Furthermore, using 300mM NaCl chloride may also create salting out effect. did the original buffer had salt ?

I totally agree with Ranjan! Doublecheck your pI and try lower and higher salt. Additives like glycerol have also been described in the literature to reduce aggregation and to stabilize certain proteins. Why not giving 5-20% glycerol a go? In addition, you can try to do a wash / on-column dialysis step (if you have some sort of affinity tag to bind the protein to a column) and run a urea gradient from 1 to 0 M over lets say 10-20 cv before eluting in your dialysis buffer. I am not an expert on protein refolding however I think many people also add arginine to assist folding. Also temperature is a factor that probably needs to be considered ... did you do your dialysis in the fridge?

I also try to avoid phosphate buffers like PBS since phosphate has a tendency to from salt precipitates. Did you check if your precipitate is your protein or just salt?

Another thought would be construct design or expression cell line.... since you have your protein in 1 M urea it may not be properly folded? Was it ever soluble without urea? Did you try different expression cell lines including extra plasmids for rare t-RNA codons?

If nothing helps the hard truth is often it is actually better to redesign the epression constructs in order to obtain a soluble protein.

Best of luck!

What is the components of Urea containing buffer? Is it PBS too? And are there any cystein residue in yr protein? and generally you have to centrifuge the protein solution before any dialysis experiment (10000-12000 rpm, 10 min at least). What is the exact protocol which you use? (I mean the duration of dialysis and its steps)

To add up the previous comments, I would suggest you to reduce urea gradually from 1M to 0M in a step gradient basis. Well, I had some problem of protein aggregation, then I was adviced to check the hydrophobicity of the protein, and use the NaCl accordingly(ionic strength enhance the hydrophobic effect which inturn precipitates your protein!). so check whether the protein really wants more salt such as 300-500mM or it only needs 50mM???

Refolding is not a simple stuff. May be you have to increase the time of the refolding, the gradient from 1M to 0M is really recomendable. Also look at the temperature, if you do not have protease in your mixture, try to do it at room temperature and then take it in 1 L water bath into cold room, overnight. If you have disulfide bonds, it is good to add beta-mercaptoethanol (toxic). I really like to express folded proteins, I would think about others heterologous organism, as Pichia pastoris (if it is the case). If you are working with membrane proteins, look at specific protocols.

Good luck!