This likely means that your imac column is acting as an ion exchanger :-) On Jan 15, 2012 2:50 PM, "Cécile Breyton" <[email protected]> wrote:
> I addition to the suggestions of checking folding/aggregation/**proteolysis, > you might also want to try lowering the NaCl concentration. Whereas I like > having some to prevent ion exchange effects on the IMAC column, I have had > the case of a protein that does not bind if NaCl is present. Binds > perfectly well without, is eluted fine with 200 mM Imidazol. This protein > does not bind at low Imidazol concentration either by the way. > > hth, > > Cécile > > Le 15/01/12 19:23, Theresa H. Hsu a écrit : > >> Hi all >> >> I have a His-tagged soluble protein (8 His residues added to 90 kDa >> protein) that do not bind to IMAC column based on flowthrough showing up >> with Western blott. Do you have suggestions to improve the binding? >> >> Binding condition is 50 mM Tris-HCl 8.0, 300 mM NaCl, 10 mM imidazole pH >> to 8.0. >> >> Thank you. >> >> Theresa >> >> > -- > Cécile Breyton > Institut de Biologie Structurale > UMR 5075 CNRS/CEA/UJF > 41, rue Jules Horowitz > 38027 Grenoble cedex 1 France > --- > Tel: +33 (0)4 38 78 30 37 > Fax: +33 (0)4 38 78 54 94 > Courriel : [email protected] > http://www.ibs.fr/groups/**membrane-and-pathogens-group/** > ssimpa/article/ssimpas-1109<http://www.ibs.fr/groups/membrane-and-pathogens-group/ssimpa/article/ssimpas-1109> >
