His-Tag:Single column prep?

Hernan Espinoza espinoza at cgl.ucsf.edu
Thu May 18 16:19:13 EST 1995


ajotsuka at rs6000.cmp.ilstu.edu (Anthony Otsuka) writes:

>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

The short answer is NO.  I have gotten some pretty good results
using metal chelating columns, but I have found that if you
want REALLY pure protein(and no imidizole, which is bad for
proteins), you should throw in an ion-exchange column.(ie DEAE,
monoQ, S sepharose).  Anyway, you can also improve the purity of
your metal chelating column fractions by including a low pH (6.8)
wash and/or a 5% imidizole wash step(s).  One other possibility is
to change the metal you are using for your affinity matrix (I got
some great results by using cobalt instead of nickel).  If you
email me : espinoza at cgl.ucsf.edu , I can fax you a protocol.
Good luck.  - Hernan




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