Abstract
The electrophoretic mobility shift assay (EMSA) determines sequence-specific protein-deoxyribonucleic acid (DNA) interactions (e.g., transcription factors with cognate regulatory elements). This assay is based on reduced electrophoretic mobilities of protein/DNA complexes through a nondenaturing polyacrylamide gel compared with unbound DNA fragments or double-stranded oligonucleotides. If multiprotein complexes bind, the migration rate slows further with each additional protein. Oligonucleotide competition and antibody binding controls provide further evidence of binding specificity and protein identity. In competition experiments, unlabeled (cold) DNA or the same oligonucleotide (being used as a probe) with wild-type or mutated sequences of binding site or other unrelated binding sequences are added to the reaction. Interactions between the binding protein and the unlabeled related sequences would decrease the band intensity of the previously shifted complexes, whereas mutated or unrelated binding sequences will not change the intensity of the previously shifted complexes (an example is shown in Fig. 1).
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© 2004 Huaman Press Inc., Totowa, NJ
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Javed, A. et al. (2004). Protein-Deoxyribonucleic Acid Interactions Linked to Gene Expression. In: Giordano, A., Romano, G. (eds) Cell Cycle Control and Dysregulation Protocols. Methods in Molecular Biology™, vol 285. Humana Press. https://doi.org/10.1385/1-59259-822-6:045
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DOI: https://doi.org/10.1385/1-59259-822-6:045
Publisher Name: Humana Press
Print ISBN: 978-0-89603-949-0
Online ISBN: 978-1-59259-822-9
eBook Packages: Springer Protocols