[Protein-analysis] HELP! protein-protein cross-linking

=?gb2312?B?sPzI8Q==?= via proteins%40net.bio.net (by baorui_ren from hotmail.com)
Mon Aug 4 21:30:59 EST 2008



From: baorui_ren from hotmail.comTo: methods from net.bio.netSubject: HELP! protein-protein cross-linkingDate: Tue, 5 Aug 2008 02:28:20 +0000


Hello, AllWe have a problem in the chemical cross-linking experiment for protein-protein interaction. We have solved a protein complex structure, and we want to confirm their interaction by cross-linking. But i am a new comer in this field, the initial test was failed. Protein A is 35kd, Protein B is 14kd, but after cross-linking the band representing 49kd is very very weak in our SDS-PAGE comparing with the inact Protein A and Protein B in thesame lane. The condition we used is : add DSS to protein sample containing protein A and protein B and incubated for 1 hour under 4 degree, and then terminated it with 1M tris for 30 min. The samples were loaded on SDS-PAGE. We have tried different concentrations of DSS (0.1mM-10mM) and protein sample (20uM-1mM). PS: from the complex structure , we have found several Arg residues in both protein were located in the interface, so we use the DSS for initial test.Protein A trends to form dimer, but we could not even got the dimer bands after cross-linking. I would like to hear any suggestion from you.Thanks!





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