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1M imidazole sounds a bit overkill, yes.
The reason why Ni-purified proteins sometimes tend to precipitate
without imidazole is that (no matter what the suppliers claim) the metal
ions leak from the column. Thus, when in solution you have a tiny amount
of metals and the His tag in you protein, the metal ions chelate the
His-tags and aggregate you protein.
The solution is to add some chelating buffer to the prep. Imidazole is
an obvious choice, citrate etc might work as well. If your not working
with a metalloprotein, you can try EDTA also. Or maybe add enough Ni to
have a 1:1 ratio of protein:metal?
Good luck!
Pirkko Heikinheimo
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Pirkko Heikinheimo
Structural Biology and Biophysics,
Institute of Biotechnology, room 4320
P. O. Box 65, Viikinkaari 1
FIN-00014 University of Helsinki, Finland
http://www.biocenter.helsinki.fi/bi/xray/pirkko
e-mail: [EMAIL PROTECTED]
phone: 358-(0)9-191 58957
gsm: 358-(0)50-354 0713
fax: 358-(0)9-191 59940
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