imidazole eluent alternative in his-tagged proteins
Dr Dave Parcej
parcej at nospam.de
Mon Nov 1 09:30:44 EST 1999
I remember an article in Elsevier's Technical Tips Online
(http://www.biomednet.com/db/tto). "Purification of a hexahistidine-tagged
protein using L-histidine as the eluent"
Steve Gort and Stanley Maloy
I must admit that i tried it but had problems at 4oC wuth the histidine
Hope this helps
In article <4wES3.8856$it.226562 at news2.rdc1.on.home.com>, "Tyson"
<tyson at canada.com> wrote:
> Herr Behrends,
> ....have you tried eluting it with imidazole at varying concentrations yet?
> ....perhaps you can do test elutions w/ increasing [imidazole]
> ...also, is the haeme group buried in the protein?...if it is, the imidazole
> probably won't reach the interior of the folded protein and will instead
> compete w/ the exposed His tag-Ni complex.
> ....if you're looking for a protocol re: C-terminal tagging & cleavage...try
> the Qiagen expression/purification system.
> viel Glück!
> Soenke Behrends <behrends at plexus.uke.uni-hamburg.de> wrote in message
> news:381b0597.6810569 at news.uni-hamburg.de...
> | Dear netters,
> | I am working on a hemoprotein where the heme is bound
> | to the protein by a histidin (and specifically by the imidazol
> | moiety). My impression and fear is, that after his-tag binding
> | to a Ni column the elution with imidazol buffer not only
> | destroys the protein column interaction but also frees
> | my protein of heme. Does anyone know of an alternative
> | way of elution (under native conditions) get the protein
> | off the column.
> | I have thought also to construct a His-tag with TEV protease
> | site and cleave it off the column, but I am forced to tag
> | the C-terminus and I have not found any vector / technique
> | to do that.
> | Thanks a lot for any hint or comment
> | Soenke
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