His Tag purification
legatek at mcmail.cis.mcmaster.ca
Wed Jan 29 13:24:42 EST 2003
On 29 Jan 2003 mjcavallin at yahoo.com.ar wrote:
> I am working with the bacterial his tag expression system from Qiagen, and am trying to purify the his-tag protein. Apparently, the recombinant protein is going into inclusion bodies, so I am trying to purify it under denaturing conditions (8 M urea). The problem is that after lysis there is a lack of the His tag, because it is not longer recognised by Ni-NTA conjugate (Qiagen) Does any one have any recommendations of how to generate a lysate without losing the His tag?
Are you sure you are losing the his tag (ie. does a western blot with
anti-His antibody no longer work)? I have had difficulties with some
amino-terminal his tagged proteins not recognising Ni-NTA agarose,
although the epitope was clearly there. If it is possible for your
protein, try moving the tag to the carboxyl terminus.
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legatek at mcmaster.ca Kyle Legate legatek at hotmail.com
Tower of Tongues:Thursday PM:10:30-11:30 EDT:http://cfmu.mcmaster.ca
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