title: Radioligand Binding Assays for Determining Dissociation Constants of Phytohormone Receptors.
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.
authors.affiliation: Department of Molecular Signal Processing, Leibniz Institute of Plant Biochemistry (IPB), Weinberg 3, 06120, Halle (Saale), Germany.
authors.forename: Antje
authors.initials: A
authors.lastname: Hellmuth
authors.affiliation: Department of Molecular Signal Processing, Leibniz Institute of Plant Biochemistry (IPB), Weinberg 3, 06120, Halle (Saale), Germany. LuzIrina.Calderon@ipb-halle.de.
authors.forename: Luz Irina A
authors.initials: LI
authors.lastname: Calderón Villalobos
mesh_terms: D029681:Arabidopsis Proteins / Q000737:chemistry; D001667:Binding, Competitive; D044783:F-Box Proteins / Q000737:chemistry; D007210:Indoleacetic Acids / Q000737:chemistry*; D007700:Kinetics; D008024:Ligands; D046912:Multiprotein Complexes / Q000737:chemistry*; D010937:Plant Growth Regulators / Q000737:chemistry*; D011869:Radioligand Assay / Q000379:methods*; D011956:Receptors, Cell Surface / Q000737:chemistry
chemical_list: D029681:Arabidopsis Proteins; D044783:F-Box Proteins; D007210:Indoleacetic Acids; D008024:Ligands; D046912:Multiprotein Complexes; D010937:Plant Growth Regulators; D011956:Receptors, Cell Surface; C501405:TIR1 protein, Arabidopsis
keywords: Binding affinities; Competition binding; Hormone receptor; Radioligand binding; Saturation binding