i am not sure this would work as his protein seems to be degraded by the n end degradation pathway. i feel like it almost needs to be expressed as a fusion protein with some stabilizing sequence
On Fri, Aug 23, 2013 at 10:08 PM, Roger Rowlett <rrowl...@colgate.edu>wrote: > 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" <ja...@graduate.org> 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 >> >> >> >
