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Why not adopt a classical purification strategy? IEX-HIC-GEC. His-tag not required. With your protein strongly basic, anion exchange seems like a likely first step. After IEX, hydrophobic interaction or salt precipitation followed by gel exclusion is normally enough for well-expressed proteins. Roger Rowlett On Aug 23, 2013 9:27 PM, "Jahan Alikhajeh" <[log in to unmask]> wrote: > Dear Friends, > > I have been trying to purify a protein (27 kDa) which has a sumo plus 6 > His-tag at N-ter giving totaly a 45 kDa protein. > This protein does not express without sumo tag and has a poly basic tail > (Arg and Lys) at its N-ter. It does polymerize at acidic pHs. > When I tried to purify it with chelating Ni-NTA, it did not bind to the > column. I thought perhaps His-tag hided somewhere in the protein and is not > accessible thus, I repeated the experiments at 8 M urea. It did not make > any difference; very low binding to the column with high amount of unbound > protein in FT. Your advice is highly appreciated. > > Regards, > Jahan > > >