Dear Heng,
Tris forms only a very weak complex with Zn2+. It is unlikely that the
problem arises from Tris. It might be simply that the complex
dissociates in the presence of high imidazol when you elute the complex
from the nickel column. Imidazol is a pretty good Zn2+ coordinating
molecule. DTT in higher concentrations (1 mM should be now problem for a
zinc finger) can also strip of a Zn2+; it is a high affinity bidental
chelator.
HTH
Guenter
Date: Sat, 22 May 2010 11:17:41 +0800
From: rh_ibp2...@hotmail.com
Subject: [ccp4bb]
To: CCP4BB@JISCMAIL.AC.UK
Dear all,
Recently, I am working on a complex which includes two protein
subunits. The interaction was based on the Zinc Finger motif of one
protein. I co-purified the complex by nickel affinity column with one
protein bearing a C terminal His tag and the other without any
affinity tags. However, the complex was disassociated when applied to
size exclusion chromatography. The buffer I use for SEC is 20mM
Tris-HCl, 150mM NaCl, 1mM DTT, 5% Glycerol, pH 7.5, whearas the buffer
I use for nickel affinity column is 50mM Na2HPO4, 10mM KH2PO4, 137mM
NaCl, 2.7mM KCl, 10% Glycerol, pH7.4. So I am wondering is it possible
for the Tris buffer to strip the Zn ions from the Zinc Finger motif of
one protein that leads to the destruction of the complex?
I will be very appreciated if anyone has some experience in such case
and would like to share with me!
Sincerely,
Heng
Institute of Biophysics,
Chinese Academy of Sciences,
Beijing 100101, China
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--
***
Priv.Doz.Dr. Guenter Fritz
Fachbereich Biologie
Sektion Naturwissenschaften
Universitaet Konstanz
http://www.biologie.uni-konstanz.de/fritz
Universitaetsstrasse 10
Postfach M665
D-78457 Konstanz
e-mail: guenter.fr...@uni-konstanz.de
Phone Office: +49-(0)7531 88 3205
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Fax: +49-(0)7531 88 2966
