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If suppression of adventitious protein binding to Ni-NTA with elevated imidazole concentration is not practical (depending on the protein you can use up to 50 mM or higher), then you might try separating the proteins using hydrophobic interaction chromatography, which is rapid and very powerful. Personally, I would try butylsepharose. Most proteins will stick to this medium at 1 M AmSO4. Gradient elution (transitioning to 0 M AmSO4) should identify conditions of rapid step gradient separation if the proteins are separable. If your protein is already highly purified, you should be able to carry out this separation on a 1 mL or 5 mL mini-column that will take only minutes on an FPLC. Cheers, -- ------------------------------------------------------------------------ Roger S. Rowlett Professor Colgate University Presidential Scholar Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: [log in to unmask] Ailong Ke wrote: > 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 >>> >>> >>> >>> -- >> >> .............. >> 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: [log in to unmask] > --------------------------------------------