《蛋白质-核酸相互作用技术》相关实验技术-EMSA of zinc finger proteins.pdfVIP

Journal of Inorganic Biochemistry 105 (2011) 569 –576 Contents lists available at ScienceDirect Journal of Inorganic Biochemistry journal h omepage: /locate/j inorgbio Electrophoretic mobility shift assay of zinc finger proteins: Competition for Zn2+ bound to Sp1 in protocols including EDTA Rajendra Kothinti a, Niloofar M. Tabatabai b, David H. Petering a,⁎ a Department of Chemistry and Biochemistry, University of Wisconsin–Milwaukee, Milwaukee, WI 53201-0413, USA b Division of Endocrinology, Metabolism and Clinical Nutrition and Kidney Disease Center, Medical College of Wisconsin, Wauwatosa, WI 53226, USA a r t i c l e i n f o a b s t r a c t Article history: The electrophoretic mobility shift assay (EMSA) offers a principal method to detect specific DNA–protein Received 25 January 2010 interactions. As commonly conducted, the reaction and electrophoresis running buffers contain large concentrations Received in revised form 18 August 2010 of EDTA. EDTA has large affinity for Zn2+ and readily competes with zinc finger peptides for Zn2+ resulting in protein Accepted 19 August 2010 unfolding. Nevertheless, EMSA is routinely used to detect zinc finger protein–DNA adducts. This paper examines the Available online 31 August 2010 chemistry that permits the detection of zinc finger–DNA complexes in the presence of EDTA, using Zn3-Sp1 and a cognate DNA binding site, GC1. Twice as much adduct was detected when the reaction was cond