While a binary binding experiment follows a straightforward and simple setup—a fluorescently labeled target, maintained at a constant concentration, is mixed with a dilution series of a non-fluorescent ligand—a ternary binding experiment involves three molecules. The third molecule in this setup can play several roles and the linked guides describe one possible assay setup in more detail:

• Competitive Binding: The third molecule binds competitively to the target, displacing the ligand.
• Additional Binding: The third molecule binds to the target-ligand complex, adding another layer of interaction.
• Mediated Interaction: The third molecule facilitates the interaction between two molecules by binding to both, forming a ternary complex (e.g., a PROTAC or molecular glue).

Although these binding assays differ in certain ways, they all share the following features:

• Only one of the molecules is fluorescently labeled.
• A high-quality binary assay should be established first before introducing a third molecule.
• For the ternary binding affinity experiment, two molecules (always including the labeled target) are kept at constant concentration, while the fluorescence signal is recorded across a serial dilution of the third interaction partner.