I would go for DTPA and acid pH.
Better than dialysis, I would use IEX and flush le bound protein with a
buffer with as low a pH as possible with 10-100 mM DTPA, then wash with
no DTPA, and elute le protein with clean (e.g. treated with Chelex) salt.
HTH,
Pr. Nadir T. Mrabet
Structural& Molecular Biochemistry
Nutrigenex - INSERM U-954
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax: +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet<at> medecine.uhp-nancy.fr
On 11/05/2010 19:12, Javier Gonzalez wrote:
Since you have only Asp and His ligands coordinating the Fe ion,
dialysis against, say, 0.1 M Citrate at pH 5 will do the trick.
Citrate will chelate the Fe(III) (if the protein has Fe(II) it will be
oxidized during dialysis due to air oxygen) avoiding precipitation of
Fe(OH)3 and the acid will protonate the protein ligands. If the
affinity is really high, adding also some guanidinium chloride
(0.5-1M) in the first dialysis step to loosen the protein a little bit
will also help.
Best,
Javier M. Gonzalez, PhD.
University of Maryland Baltimore
Department of Pharmaceutical Sciences
X-Ray Crystallography Shared Service (UMXSS)
20 Penn St., HSFII, Rm 514
Phone/Fax: 410-7061124/410-7060886
21201 Baltimore, MD
http://www2.pharmacy.umaryland.edu/psc/xray/
On Tue, May 11, 2010 at 12:26 PM, Roger Rowlett <rrowl...@colgate.edu
<mailto:rrowl...@colgate.edu>> wrote:
I would try dialyzing against a solution with 1,10-phenanthroline,
1,10-phenanthroline-2-carboxylate, or pyridine-2,6-dicarboxylate.
Removal of metals from proteins is often not just dissociative,
but requires the associative interaction of a chelating agent.
Which one works is often empirical.
Cheers.
On 5/11/2010 11:11 AM, Wenguang LIANG wrote:
Dear all,
I have a protein which binds iron with two D and two H as active
site. I have tried to extract the iron by dialysis. First, 20mM
tris, pH7.5, 150mM NaCl, 20EDTA, 10mM Na2S2O3, O/N. Then,
followed by dialysis with 20mM tris, pH7.5, 150mM NaCl, 1EDTA O/N
to remove the EDTA and Na2S2O3. However, after dialysis, the
density of the Iron is still in the protein.
Could anybody please give me some suggestion on how to extract
Iron out of the protein? Or I can just use Chelex-100 to extract
it instead of Iron.
Thank you very much for help,
Best wishes,
Vinson Liang
--
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Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
tel: (315)-228-7245
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fax: (315)-228-7935
email: rrowl...@colgate.edu <mailto:rrowl...@colgate.edu>
