His6-tagged protein purification

Wolfgang Schechinger Wolfgang.Schechinger at med.uni-tuebingen.de
Wed Feb 23 16:26:50 EST 2000


Angela, this looks as if the His tag somehow is buried under these 
conditions. Do you have the possibility to insert a spacer between 
your protein and the tag or to move it to the other terminus?
You could check the protein's physical chemistry using Antheprot 
(http://ibcp.fr/ANTHEPROT/) for finding out the reason why.

Good luck, 

Wolfgang


> From:          a.manning at ualberta.ca (Angela Manning)
> Subject:       His6-tagged protein purification
> Date:          Wed, 23 Feb 2000 12:03:17 -0700
> Organization:  Genetics U of Alberta
> To:            methods at hgmp.mrc.ac.uk

> I am trying to purify a bacterially exressed His6 tagged protein on
> a Probond column under native conditions.  I have purified the
> protein under denaturing conditions, however, the protein is
> "completely" insoluble after denaturation.  So, I want to purify the
> soluble fraction under native conditions.  However, when I do this
> the protein is contaminated with lots of other proteins, even after
> multiple washes.  I have also tried washing in a low concentration
> (40mM) of imidazole but my protein largely washed off under these
> conditions. Does anyone have any suggestions as to how I might
> purify this protein?  Thank you!
> 
> -- 
> Angela Manning       a.manning at ualberta.ca
> Dept of Biological Sciences
> University of Alberta
> Edmonton, AB Canada
> T6G 2H3
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Dr. Wolfgang Schechinger, Dept. of Pathobiochemistry
University of Tuebingen, Germany
email: wolfgang.schechinger at med.uni-tuebingen.de 
wwWait: http://www.medizin.uni-tuebingen.de/~wgschech/start.htm
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