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opinion - denatured protein purification (Feb/08/2006 )

I have a protein which is very insoluble. I have solubilised the pellet using 8M urea. I have also dialysed the Urea to final concentration of 100 mM exhaustively. I am trying different refolding conditions and trying to find out the most optimum. Unfortunately no success until now. Now I am thinking of purifying the protein in the denatured state and then try to refold it later. Could you please let me know if the purification of the denatured protein is a good idea and if it would create any problems with Ni NTA columns. Also could you tell me about the salt concentration that I should use for purification of my his tag protein. Thank you,


I used the QIAexpressionist handbook (I have attached it).

Page 90 describes the method used for NiNTA purification under denaturing conditions with the Buffer components listed on page 113. But read it all as from page 63 there are good tips to help decide on some purification methods and they discuss issues of refolding etc.

However, as you've gone to the trouble dialysing the urea to 100mM and your proteins stays in solution then dont used 8M urea in the buffer just use 100mM (My be obvious but wouldnt want you to do it by mistake).

You will have to choose whether you want to use the imidazole gradient or a pH gradient. I found that the pH gradient worked reasonably ok two of my proteins (except for a few contaminating proteins inone prep. I repeated the purification using 20mM imidazole to try to inhibit the binding but it didnt work for me).