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Shift-Western Blotting: Separate Analysis of Protein and DNA from Protein–DNA Complexes

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Western Blotting

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1312))

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.

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Acknowledgement

I want to express my special thanks to Stephen Demczuk and Björn Vennström, with whom I had originally developed the shift-western blotting method, and Piero Carninci for his help to make this protocol possible. I further want to thank Mitch Dushay for critically reading the manuscript and his suggestions to improve the text. This work was supported by a research grant from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) to the RIKEN Center for Life Science Technologies (No. 95).

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Authors and Affiliations

  1. 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

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  1. Matthias Harbers
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Correspondence to Matthias Harbers .

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Editors and Affiliations

  1. Department of Medicine, University of Oklahoma, Arthritis and Clinical Immunology, Oklahoma Medical Research Foundation, Health Sciences Center, Department of Veterans, Oklahoma City, Oklahoma, USA

    Biji T. Kurien

  2. Department of Medicine, Arthritis and Clinical Immunology, Oklahoma Medical Research Foundation, Health Sciences Center, Department of Veterans Affairs Medical Center, Oklahoma City, Oklahoma, USA

    R. Hal Scofield

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Harbers, M. (2015). Shift-Western Blotting: Separate Analysis of Protein and DNA from Protein–DNA Complexes. In: Kurien, B., Scofield, R. (eds) Western Blotting. Methods in Molecular Biology, vol 1312. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2694-7_36

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  • DOI: https://doi.org/10.1007/978-1-4939-2694-7_36

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2693-0

  • Online ISBN: 978-1-4939-2694-7

  • eBook Packages: Springer Protocols

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