Re: [ccp4bb] Ni-NTA purifications contaminated with E coli Glucosamine-fructose-6-phosphate aminotransferase

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Ni-NTA purifications contaminated with E coli Glucosamine-fructose-6-phosphate aminotransferase
From: Ailong Ke ak425 {- at -} CORNELL {- dot -} EDU
Date: 2007-11-02

Next message:
Subject: Re: Ni-NTA purifications contaminated with E coli Glucosamine-fructose-6-phosphate aminotransferase
From: Ailong Ke ak425 {- at -} CORNELL {- dot -} EDU
Date: 2007-11-02

Subject: Re: Ni-NTA purifications contaminated with E coli Glucosamine-fructose-6-phosphate aminotransferase
From: Christian Biertumpfel biertumpfelc {- at -} NIDDK {- dot -} NIH {- dot -} GOV
Date: 2007-11-02

Hi Ailong,
Use a high salt concentration in your lysis buffer (0.5-1 M NaCl). You
could also try to add some imidazole (e.g. 30 mM). This removes a lot of
unspecific binding to the Ni-column. After applying the lysate I usually
wash with 60 mM imidazole for at least 20 CV and then I elute with
300 mM imidazole. Another option is to try different resins (Ni-NTA vs
Ni-Sepharose)

Hope this helps,
christian

Ailong Ke wrote:
>> Hello,
>
>
> We are trying to purify an N-terminal His6-tagged protein from E. coli,
> and the prep was contaminated with the E coli
> Glucosamine-fructose-6-phosphate aminotransferase, which co-purifies
> with my protein of interest in subsequent ion-exchange and sizing
> columns. This protein appears to be a common contamination in the Ni-NTA
> purifications. Does anyone have tricks to get rid of it? Thanks.
>
> Ailong
>
>
>

_______________________________________________________________________

Dr. Christian BiertÃ¼mpfel
Laboratory of Molecular Biology

NIDDK/National Institutes of Health phone: +1 301 402 4647
9000 Rockville Pike, Bldg. 5, Rm. B1-03 fax: +1 301 496 0201
Bethesda, MD 20892-0580
USA
_______________________________________________________________________

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Ni-NTA purifications contaminated with E coli Glucosamine-fructose-6-phosphate aminotransferase
From: Ailong Ke ak425 {- at -} CORNELL {- dot -} EDU
Date: 2007-11-02

Next message:
Subject: Re: Ni-NTA purifications contaminated with E coli Glucosamine-fructose-6-phosphate aminotransferase
From: Ailong Ke ak425 {- at -} CORNELL {- dot -} EDU
Date: 2007-11-02