Hi Tasada, do I understand it correctly: you have purified your protein in a denatured state (with urea) and after elution from Ni-NTA you want to dialyse out the urea to refold it? If, yes, I can imagine, that your protein is simply precipitating/aggregating, as it does not refold correctly, which is why you canno detect it on SDS-PAGE anymore. You should try other refolding conditions (read e.g. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC517725/), or even another denaturing agent, e.g. GuHCl. Another possibility is rapid dilution, where you do not dialyse out the denaturant slowly, but dilute your denatured protein quickly into a large volume of buffer. This is i a way "more physiological", as it resembles more what happens in vivo (e.g. with the hemolysin system in E. coli, or other secreted proteins). For testing many refolding conditions there are 3 commercial kits available (called iFold from Merck). We have prepared them for ourselves in our lab to minimize the costs - the manuals give you a good idea of possible methods and refolding buffers. Good luck, Magdalena

Hi Tasada, do I understand it correctly: you have purified your protein in a denatured state (with urea) and after elution from Ni-NTA you want to dialyse out the urea to refold it? If, yes, I can imagine, that your protein is simply precipitating/aggregating, as it does not refold correctly, which is why you canno detect it on SDS-PAGE anymore. You should try other refolding conditions (read e.g. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC517725/), or even another denaturing agent, e.g. GuHCl. Another possibility is rapid dilution, where you do not dialyse out the denaturant slowly, but dilute your denatured protein quickly into a large volume of buffer. This is i a way "more physiological", as it resembles more what happens in vivo (e.g. with the hemolysin system in E. coli, or other secreted proteins). For testing many refolding conditions there are 3 commercial kits available (called iFold from Merck). We have prepared them for ourselves in our lab to minimize the costs - the manuals give you a good idea of possible methods and refolding buffers. Good luck, Magdalena

I will answer the second part of the question... Proteins are usually run on SDS-PAGE and the cut bands are processed and injected to the animal for antibody productions. The Acrylamide polymers work as adjuvants here and polymeric acrylamide is non-toxic..

For changing buffer or desulting the protein, you can use desulting colums such as G-10. This would be very faster (10_30 min) than dialysing the protein over a long period of time, 12-24 hours.

I think that you should size exclusion chromatography to such as on a G-10 column to change buffer. If it is a protease that is cleaving your protein then you may want to add protease inhibitor cocktail to your buffer. Or purify your protein in a size exclusion column such as sephacryl S-200. That way you have pure protein and you may be able to store it for longer time.

On size exclusion you will have disaster of aggregates will stay at the beginning of the column in case of switching from 8M urea into native conditions. Please don't do it. Please...

The advices I voted up of Magdalena Schacherl and Santosh Kumar are excellent. You have a very nicely purified protein ready to run on the gel and slice it of. Don't try to overdo with your successful purification Please... ;)

If aggregation is an issue then size exclusion would be tricky. On the other hand how much of protein one can purify in a gel is a concern.

Thanks all for your valuable answers. I think I would go with Jan Piwowarski.

I am pretty sure you can give protein in urea, up to 1M for Ab production. I would check with companies that make the Abs. I also like the SDS PAGE/ cutting out of band technique. Thumbs up for that answer.

I am not sure about what it is happening with your protein. During your dialysis are you able to see aggregation of the protein? or you obtain a clear solution but without protein in it?...the causes of these two possibilities are different. If you obtain precipitation then you have towork on the refolding procedure (performing it by steps, with redox, aminoacids, etc.). If your protein simply disappears, you can consider if you are really using the correct dialysis membrane pore. I see that your protein is small and you didn´t say anything about the aparent Mw therefore, if you have a monomer and you are using a dialysis membrane with a big pore, you are loosing your protein. Regarding isolation of the protein from the gel, I have done several times with good results

as Magdalena and others have suggested, when you remove the urea, your protein is likely precipitating, probably on the column, as you go to 0 M urea. So I would keep your protein in high urea for the Ni NTA, you should not loose it. Then do the refolding using the refold kits. Once you know which buffer works best, you can try doing the refolding on the NI NTA resin. I typically go from buffer with 15mM imidazole, 300mM NaCl in 6M urea to 2M, to 1M with the washes and use about 5 column volumes. Then I do a wash of 3 column volumes using your refold buffer with 15mM imidazlole. and then I elute with 0.5M imidazlole in your refolding buffer.

If you do dialysis after this, and loose the protein, ie it precipitates in the cassette, then you may have to lower the concentration of your protein even befor dialysis, as once it starts to precipitate, the microcrystals help more protein precipitate.