Abstract
In receptor–ligand interactions, dissociation constants provide a key parameter for characterizing binding. Here, we describe filter-based radioligand binding assays at equilibrium, either varying ligand concentrations up to receptor saturation or outcompeting ligand from its receptor with increasing concentrations of ligand analogue. Using the auxin coreceptor system, we illustrate how to use a saturation binding assay to determine the apparent dissociation constant (K ′D ) for the formation of a ternary TIR1–auxin–AUX/IAA complex. Also, we show how to determine the inhibitory constant (K i) for auxin binding by the coreceptor complex via a competition binding assay. These assays can be applied broadly to characterize a one-site binding reaction of a hormone to its receptor.
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Acknowledgments
This work was supported by the IPB core funding from the Leibniz Association and the Deutsche Forschungsgemeinschaft (DFG) through the research grant CA716/2-1 to L.I.A.C.V.
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Hellmuth, A., Calderón Villalobos, L.I.A. (2016). Radioligand Binding Assays for Determining Dissociation Constants of Phytohormone Receptors. In: Lois, L., Matthiesen, R. (eds) Plant Proteostasis. Methods in Molecular Biology, vol 1450. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3759-2_3
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DOI: https://doi.org/10.1007/978-1-4939-3759-2_3
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