Thanks for the suggestions, JJ.
The supernatant was loaded onto Ni-NTA in the presence of 300 mM NaCl
and 5 mM imidazole, washed with 20 mM imidazole (could test higher
concentration...), then eluted with 300 mM imidazole. So I don't
think non-specific binding is a problem. We got 90% purity off the
Ni-NTA. But for this particular protein , the
Glucosamine-fructose-6-phosphate aminotransferase just wouldn't go
away in subsequent purifications (which may imply physical
interactions?).
I guess I was wondering if other people had similar problems, and if
there exists a trick similar to using ATP to remove GroEL
contamination.
Ailong
Provided your proteins don't stick to each other through exposed
hydrophobic surfaces
then sodium chloride could be used to separate the contaminants more
efficiently prior
to NiNTA purification. You should wash with 30-40 (!!!!) column
volumes in 20-30mM
imidazole to remove non-specific contaminants. Conslut your manual (Ni-resin)
to find out limitations due the presence of higher salt concentraitons.
JJ
On Nov 2, 2007, at 2:42 PM, 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
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..............
Joachim Jaeger, DPhil
Center for Medical Sciences, Rm.2009
NYS-DOH, Wadsworth Center
Albany, New York 12201-0509
Tel: +1 518 408-2225 Fax: +1 518 402-2633
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Ailong Ke, Ph.D.
Assistant Professor
Department of Molecular Biology and Genetics
Cornell University
251 Biotechnology Building
Ithaca, NY, 14853
tel: 607-255-3945
fax: 607-255-6249
email: [EMAIL PROTECTED]
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