Abstract
The recognition of specific DNA sequences by proteins is crucial to fundamental biological processes such as DNA replication, transcription, and gene regulation. The technique of surface plasmon resonance (SPR) is ideally suited for the measurement of these interactions because it is quantitative, simple to implement, reproducible, can be automated, and requires very little sample. This typically involves the direct capture of biotinylated DNA to a streptavidin (SA) chip before flowing over the protein of interest and monitoring the interaction. However, once the DNA has been immobilized on the chip, it cannot be removed without damaging the chip surface. Moreover, if the protein–DNA interaction is strong, then it may not be possible to remove the protein from the DNA without damaging the chip surface. Given that the chips are costly, this will limit the number of samples that can be tested. Therefore, we have developed a Reusable DNA Capture Technology, or ReDCaT chip, that enables a single streptavidin chip to be used multiple times making the technique simple, quick, and cost effective. The general steps to prepare the ReDCaT chip, run a simple binding experiment, and analysis of data will be described in detail. Some additional applications will also be introduced.
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Acknowledgments
The work was carried out using the John Innes Centre Biophysical Analysis Facility with funding from the Biotechnology and Biological Sciences Research Council (UK) Institute Strategic Programme Grant BB/P012523/1. We would like to thank Julia Mundy for critically reading the chapter.
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Stevenson, C.E.M., Lawson, D.M. (2021). Analysis of Protein–DNA Interactions Using Surface Plasmon Resonance and a ReDCaT Chip. In: Daviter, T., Johnson, C.M., McLaughlin, S.H., Williams, M.A. (eds) Protein-Ligand Interactions. Methods in Molecular Biology, vol 2263. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1197-5_17
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DOI: https://doi.org/10.1007/978-1-0716-1197-5_17
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