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getting zinc into a zinc finger

pupcia pupcia at my-deja.com
Fri Jan 28 10:03:03 EST 2000

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 concentrating my 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.  Does anybody have
tips on things to watch for or point to an explicit protocol?



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