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Re: [ccp4bb] Off topic: His-tag purification |
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CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999Subject: Re: Off topic: His-tag purification From: Cécile Breyton Cecile {- dot -} Breyton {- at -} IBS {- dot -} FR Date: 2012-01-15 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 : Cecile.Breyton@ibs.fr http://www.ibs.fr/groups/membrane-and-pathogens-group/ssimpa/article/ssimpas-1109 CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999 |
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