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
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Tel: +33 (0)4 38 78 30 37
Fax: +33 (0)4 38 78 54 94
Courriel : cecile.brey...@ibs.fr
http://www.ibs.fr/groups/membrane-and-pathogens-group/ssimpa/article/ssimpas-1109
