I am trying to put zinc into a LIM domain zinc finger in preparation for
NMR structural studies. I am starting from lyophilized protein. I
have been doing a literature search and but I haven't discovered a
really explicit protocol.
Most of the published papers say that I should mix my protein in acid
conditions with a mole equivalency of my metal and then neutralize with
a base to the pH I want. Some papers perform this under anaerobic
conditions, others perform it only in the presence of reducing agents
(bME, DDT). The sources of zinc are either ZnCl2 or ZnSO4. The methods
of purifying/concentrating the metallated protein range from dialysis in
the presence of dilute metal chelator to simple ultrafiltration.
I am looking for comments on what procedure to use. Has anyone done this
who can tell me what to do/not to do?
Thanks,
Tine
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