Hello Haitham,

1. If the tag of your protein is MBP+ His tag, it is definitely better to use IMAC to remove the processed tag after Tev protease treatment. First, the resin is cheaper. Second if you used adsorption to amylose followed by elution with maltose, it is next to impossible to remove the maltose bound to the MBP tag and the latter will never bind to the amylose column. If you used IMAC for the purification of the tagged protein, all you have to do is restore the pH (if eluting at low pH) or remove imidazole by GF or dialysis (if eluting with imidazole). Upon removing the cleaved tag with a second passage on Ni-NTA, it is advisable to add some imidazole (~20 mM) to the buffer, since  some detagged proteins will stick to the resin at 0 mM imidazole.

2. I am not sure I understand your second question. However, if  you are asking whether  a desalting column is OK to remove urea and refold you protein, I would say no. In order to perform gel filtration, your protein has to be loaded in a relatively small volume, hence at a relatively high concentration; thus it will all the more likely to precipitate and clog your column upon removal of urea.

Thanks Pierre,

Yes that what I meant by the 2nd question, and I understand your question well, but then do the ethanol precipitation could be helpful in such a case? and does it fit with the downstream immunization process? 

Is your Histag-MBP-ProteinOfInterest already in Urea? In that case you have to use IMAC, since your MBP will be also denatured (partially or fully based on your Urea concentration) and incapable of binding to amylose resins.

Thanks Ramesh,

This is a good tip, Urea will be excluded from the amylose resin cell lysis buffer.