May be GST interacts with your protein. Is your protein having Cysteine residues (specially in the active site). What is the function and size of your protein. I was facing the same problem.
On 1/14/12, AllisonH <[email protected]> wrote: > On 13/01/2012 12:29 PM, Phelan, Paul J. wrote: >> I am trying to purify a protein, starting with a fusion that is >> double-tagged with GST and poly-His, and my protein of interest also has >> an n-terminal poly-His tag of its own. So the fusion protein is >> polyHis-GST-polyHis(Protein). An initial step with glutathione Sepharose >> works just fine, the fusion protein binds and is eluted with 10 mM >> glutathione. Digestion of the fusion protein (with His-tagged TEV >> protease) also works, and gives me the polyHis-GST tag, plus >> polyHis(Protein). BUT - when I re-run the digested protein over >> glutathione Sepharose, the polyHis-GST is not removed, and just passes >> through the column, along with the polyHis(Protein). Has anyone else seen >> a problem with GST from a digested fusion protein not binding to >> glutathione Sepharose? Could it be that the GST still has glutathione >> tightly bound to it? (The fusion protein is digested by extensive >> dialysis with TEV protease). Could it be that the polyHis-GST is not >> behaving the same as non-tagged > GST? This seems doubtful, since the fusion protein binds to glutathione > Sepharose in the first step. Before anyone suggests using a Nickel resin to > remove the polyHis-GST, that's also a problem, since my protein of interest > (and also the TEV protease) is His-tagged too. >> >> Any suggestions/insights would be welcome! >> >> Paul Phelan >> Tufts University >> Boston >> > > If you have a protein that already has an internal His site you should > be able to purify it on a nickel column without any tags. In fact, by > adding a second His tag you probably will not be able to elute it > easily. (I had exactly this problem a few years back). > Allison > _______________________________________________ > Methods mailing list > [email protected] > http://www.bio.net/biomail/listinfo/methods > -- With regards, Krishn Pratap Singh Dept of Biochemistry R.M.R.I.M.S. (I.C.M.R.) Agamkuan, Patna-800004 Phone no: 09304763794 _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
