His-Tag:Single column prep?

Dr. Robert Bateman rbateman at wave.st.usm.edu
Fri May 19 10:42:51 EST 1995


Hernan Espinoza (espinoza at cgl.ucsf.edu) wrote:
: 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

On the topic of using other metals, does anyone know what the metal is in 
the new Clonetech TALON resins? They won't tell.

Bob Bateman



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