In addition to all good recommendations I would add the following: load your protein to the whatever-Ni column then wash it with urea. 1 to 4 M urea will not, in most cases, destroy your protein but will disrupt the hydrophobic interaction between contaminant and your protein in case if the contaminant is binding not to Ni column but to your protein. My two cents. Vaheh Oganesyan, Ph.D. MedImmune, Inc. Phone: 1-301-398-5851 Facsimile: 1-301-398-8851
To the extent this electronic communication or any of its attachments contain information that is not in the public domain, such information is considered by MedImmune to be confidential and proprietary, and expected to be used only by the individual(s) for whom it is intended. If you have received this electronic communication in error, please reply to the sender advising of the error in transmission and delete the original message and any accompanying documents from your system immediately, without copying, reviewing or otherwise using them for any purpose. Thank you for your cooperation. -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] Behalf Of Ailong Ke Sent: Friday, November 02, 2007 3:53 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Ni-NTA purifications contaminated with E coli Glucosamine-fructose-6-phosphate aminotransferase 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: [EMAIL PROTECTED] --------------------------------------------
