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: ya...@moon.ibp.ac.cn <mailto:ya...@moon.ibp.ac.cn>
We will either find a way or make one.
