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 Biertümpfel 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 _______________________________________________________________________
