title: Shift-Western Blotting: Separate Analysis of Protein and DNA from Protein-DNA Complexes.
abstract: The electrophoretic mobility shift assay (EMSA) is the most frequently used experiment for studying protein-DNA interactions and to identify DNA-binding proteins. Protein-DNA complexes formed during EMSA experiments can be further analyzed by shift-western blotting, where the protein and DNA components contained in a polyacrylamide gel are transferred to stacked membranes: First a nitrocellulose membrane retains the proteins while double-stranded DNA passes through the nitrocellulose membrane and binds only to a charged membrane placed below. Immobilized proteins can then be stained with specific antibodies while the DNA can be detected by a radioactive label or a nonradioactive detection system. Shift-western blotting can overcome many limitations of supershift experiments and allows for the analysis of complex protein-DNA complexes containing multiple protein factors. Moreover, proteins and/or DNA may be recovered from membranes after the blotting step for further analysis by other means.
authors.affiliation: Division of Genomic Technologies, RIKEN Center for Life Science Technologies, RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan, matthias.harbers@riken.jp.
authors.forename: Matthias
authors.initials: M
authors.lastname: Harbers
mesh_terms: D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D002417:Cattle; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015342:DNA Probes / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D006801:Humans; D051379:Mice; D051381:Rats
chemical_list: D015342:DNA Probes; D004268:DNA-Binding Proteins; D004247:DNA