Abstract
Limited information exists for the binding specificities of many important transcription factors. To address this, we have previously developed a microwell-based assay for directly measuring the affinity of DNA-protein binding interactions. We describe here the detailed protocol for determining sequence specificities of DNA-binding proteins using this assay. The described method is rapid; after preparation of the reagents, the assay can be run in a single day, and its throughput can be increased further by automation. The method is quantitative but requires prior knowledge of one high-affinity binding site for the protein of interest. The protocol can be adapted for determining the effect of protein modifications and protein-protein interactions on DNA-binding specificity, and for engineering proteins with new DNA-binding specificities. In addition, the method is suitable for high-throughput screening to identify proteins or small molecules that modulate protein-DNA binding interactions.
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Acknowledgements
We thank H. Clevers for constructs and M. Björklund and A. Oinas for reviewing the protocol. This work was supported by the Translational Genome-Scale Biology Center of Excellence of the Academy of Finland, the Regulatory Genomics specific targeted research project of the European Union, Biocentrum Helsinki, the University of Helsinki, the Sigrid Juselius Foundation, the Magnus Ehrnrooth Foundation, the Jenny and Antti Wihuri Foundation and the Finnish Cancer Research Organizations.
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Hallikas, O., Taipale, J. High-throughput assay for determining specificity and affinity of protein-DNA binding interactions. Nat Protoc 1, 215–222 (2006). https://doi.org/10.1038/nprot.2006.33
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DOI: https://doi.org/10.1038/nprot.2006.33
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