《蛋白质-核酸相互作用技术》相关实验技术-Principles and problems of the electrophoretic mobility shift assay.pdfVIP

Journal of Pharmacological and Toxicological Methods 63 (2011) 7 –14 Contents lists available at ScienceDirect Journal of Pharmacological and Toxicological Methods journal h omepage: /locate/j pharmtox Original article Principles and problems of the electrophoretic mobility shift assay Neil S. Holden a,⁎, Claire E. Tacon b a Department of Cell Biology and Anatomy, Airway Infl ammation Research Group, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4N1 b Department of Physiology and Pharmacology, Airway Infl ammation Research Group, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4N1 a r t i c l e i n f o a b s t r a c t Article history: Introduction: The electrophoretic mobility shift assay (EMSA) is classically used to detect DNA binding Received 31 December 2009 proteins, the tenet of the EMSA is that DNA with protein bound, migrates through a polyacrylamide gel more Accepted 11 March 2010 slowly than the corresponding free unbound DNA. Methods: The classical EMSA protocol has 4 major steps: 1) The isolation of proteins from cells. Since the vast majority of active DNA binding proteins are present within Keywords: the nucleus, a sequential membrane lysis protocol is used which yields purified nuclear protein. 2) Manufacture EMSA 32 and radiolabelling of the DNA probe. Phosphorous 32 (