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Dear Mr. Fritz, Yes, the protein is not an E.coli protein! Instead, it was cloned from a virus. And since it was a nonstructural viral protein, I thought it might be appropiate to treat it as eukaryotic proteins. E.coli system was quite different from eukaryotic ones, hence I was quite cautious about the ICP-ES result and trying to confirm it via alternative method. Thanks very much for mentioning the examples which suggested that Fe might be contaminants. Indeed, when I cut the protein in two parts (still with MBP) and test them via ICP-ES again, Fe became negligible in both and Zn stoichiometry increaed to 1:1 in the C-terminal part. The result lead me to focus on Zn instead of Fe. But I still want to confirm the idea. Matallo biochemistry was exactly what I dreamed to do. Sincerely, Xuan Yang 2007/8/6 Guenter Fritz <[log in to unmask]> > Hi Xuan, > I guess your protein is not an E.coli protein. There are several examples > that eukaryotic Zn-proteins expressed in E.coli contain Fe instead of Zn. I > am sceptic whether IMAC with different metal ions will give the solution of > the problem. If you really want to get information on the metal ion binding > properties you will have to do some matallo biochemistry: preparing apo > protein, reconstitution with metal ions, UV-Vis spectroscopy, EPR would be > great, ... > > Dear Sir or Madam, >> The ICP-ES results indicated that 1 molar my protein purified from E.coli >> Origami(DE3) contained about a half molar Zinc and nearly a quarter molar >> Iron (whether II or III was not available). The protein carried a MBP tag on >> the N-terminal and the situation was similar with or without His tag at the >> C terminal. I want to determine whether my protein really bind Zinc or Iron. >> Does anyone have any experience about such problems? >> Specifically, now I want to compare the binding efficiency on various >> IMAC, i.e. 50mM ZnSO4, FeSO4, Fe2(SO4)3, NiSO4(control), or CuSO4(control). >> However, considering the instability of Fe(II) in solution, the design >> still seemed problematic. >> Sincerely, >> Xuan Yang >> National Laboratory of Biomacromolecules and >> Center for Infection and Immunity, >> Institute of Biophysics, >> Chinese Academy of Sciences, >> Room 1617, 15 DaTun Road,Chaoyang District, >> Beijing, China, 100101 >> Tel: 86-10-64884329 >> Academic email: [log in to unmask] <mailto:[log in to unmask]> >> We will either find a way or make one. >> >> > >