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Re: [ccp4bb] Ni-NTA purifications contaminated with E coli Glucosamine-fructose-6-phosphate aminotransferase |
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CCP4bb navigationCCP4bb <-- 2007 <-- November 2007 <-- 02 November 2007Subject: 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 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 >> >> >> >>-- >> > >.............. >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 > .-. .-. .-. .-. .-. .-. .-. .-. .-. >/ X / X / X / X / > / X / X / X / X / >`-' `-' `-' `-' `-' `-' `-' `-' `-' > > > > > >IMPORTANT NOTICE: This e-mail and any attachments may contain >confidential or sensitive information which is, or may be, legally >privileged or otherwise protected by law from further disclosure. It >is intended only for the addressee. If you received this in error or >from someone who was not authorized to send it to you, please do not >distribute, copy or use it or any attachments. Please notify the >sender immediately by reply e-mail and delete this from your >system. Thank you for your cooperation. -- -------------------------------------------- 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: ailong.ke@cornell.edu -------------------------------------------- CCP4bb navigationCCP4bb <-- 2007 <-- November 2007 <-- 02 November 2007 |
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