What is the best method to refold a recombinant protein purified under denaturing condition which is to be used for immunization?

Could you explain why you use 6M Urea in the recombinant protein preparation? Is it produced as inclusion bodies and aggregates? If so you could solubilise your protein in other solution as Buffer (PBS Or TBS or Acetate containing salt as Nacl 20 to 100 mM and with a pH different of the pHi isoelectric pH of your protein) ) with detergent eg: SDS 0.1 to 0.25% or Triton X100 0.1 to 0.25% or stabilisant as Glycerol or sucrose 10% or other solubilsant as 6M guanidine/Hcl instead of 8M Urea (may be the protein stay soluble after dialysis to eliminate this guanidum buffer.

However and in my view the refolding is not necessary in immunization.

You could realize the immunization with protein aggregates adding freund adjuvant or SDS-proteins or Triton-proteins with freund adjuvant.

I believe that many denaturated proteins have best and strong antigenicity than their native form and produced better immune serum in animals.

In some cases authors denaturate their proteins to enhance immunogenicity and probably expose more epitopes.

But we should be careful in all cases; the immune response depends of the kind, nature and the primary sequence and structure of the protein of interest.

Good work

Best regards

pH is an important factor. You may try different pH buffer for dialysis. Another common practice is adding Arg or trehalose. The concentration of a protein is also an important factor. The lower the better. In this sense, dilution is a more convenient method than dialysis.

Because you want to use the protein to generate antibody, you may not need to refold it. Depending on your experiment, denatured protein can be a better antigen. Western blot is an example. Unless you need conformational antibody, it is not necessary to refold your protein.

Another point is that refolding and solubilization of a protein can be different things. If you only need to get your protein solubilized, you can try some extreme conditions, such as high pH, add SDS or other detergents or even organic solvents.

If you have troubles to get this protein done, why not try to use peptides to generate the antibody?

Cydi from EZBiolab

Thank you Mr. Marzouki and Cydi Longering. Refolding the protein is not my actual concern. I have purified the his tagged protein on Ni-NTA column using 8M urea by varying the pH. I want to remove urea from the eluted fractions without precipitating the protein as injecting the aggregated proteins into mice is not convenient.

You have already purified the tagged protein on Ni-NTA column using 8M urea by varying the pH

You could use Centricon system

You concentrate the protein fractions by ultra-filtration on UM05 (MW exclusion 5 kDA) or PM10 (MW exclusion 10 kDA) membranes with centrifugation These membrane filter UM05 or PM10 (named Centricon) are commercially available, and after spin the protein is not filtrated on the contrary it is concentrated in its buffer (8M urea) After concentration you may mix with freund adjuvant and inject to animals

Good success

Thank you Mr. Marzouki