A competitive binding assay typically measures the binding of a labeled ligand to a target protein in the presence of a second, competing, but unlabeled ligand. This assay can be used to assess qualitative binding information as well as relative affinities of two or more molecules for one target. It can also be used to quantify high-affinity interactions that are difficult to measure directly.

The main requirement for this assay is that the given molecules bind to the same site at the target protein and the tracer is fluorescently labeled. The unlabeled competitor then competes to be bound to the same site as the labeled molecule. This assay yields an EC₅₀, the concentration that displaces 50% of the labeled molecule from the interaction site. The affinity of the non-labeled ligand for the protein can then be calculated. In case you are new to competition assays, feel free to contact the NanoTemper Technologies Support Team (NanoTemper Technologies Support) for help with setting up competitive binding assays.