His-Tag:Single column prep?

zinc zinc at biochem-49.med.utah.edu
Mon May 15 17:46:23 EST 1995


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In article <1995May15.212901.16374 at rs6000.cmp.ilstu.edu>,
Anthony Otsuka <ajotsuka at rs6000.cmp.ilstu.edu> wrote:
>We have been using pRSET vectors (Invitrogen) for expressing
>proteins.  During purification over nickel chelating columns,
>we see a considerable amount of background protein binding
>(other metal-binding proteins?).  Has anyone been able to
>achieve >90% purity by using the single column?  Any advice
>would be appreciated.  Thanks, A. Otsuka

although these might be metal binding proteins i doubt that is why
they are sticking to the column.  most of the background seems to be
from proteins that stick to hydrophobic interactions resulting from
the high [NaCl] used in the buffers (0.5M).  usually a run over some
ion exchange resin is sufficient to complete the purification.

- -pjf



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-- 
patrick finerty         --      finerty at msscc.med.utah.edu
                                pfinerty at nyx10.cs.du.edu
U of Utah biochem grad student/slave in the Bass lab
easily found at (801) 585-3110 almost anytime.   rm 207 wintrobe.



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