Date: Sat, 22 May 2010 11:17:41 +0800
From:
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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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