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 > > ------------------------------------------------------------------------ > 搜索本应是彩色的,快来体验新一代搜索引擎-必应,精美图片每天换哦! 立即试 > 用！ <http://cn.bing.com/?form=CRMADS> > ------------------------------------------------------------------------ > 聊天+搜索+邮箱 想要轻松出游,手机MSN帮你搞定! 立刻下载！ > <http://3g.msn.cn/>
-- *********************************** 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 Phone Lab : +49-(0)7531 88 3733 Fax: +49-(0)7531 88 2966
