How to perform a binding affinity assay?
FIDA is a fluorescence-based technique, hence for a binding affinity assay we need a protein with intrinsic fluorescence or a fluorophore on it. This is what we call the indicator and allows us to observe the interaction between an indicator and a binding partner. The binding partner is what we call the analyte. During a binding assay, the indicator is kept at a constant concentration and the analyte is titrated in at increasing concentrations. The analyte concentration should be high enough to reach a plateau on a binding curve to ensure full complex formation. This would allow you to extract the size of the indicator alone, the size of the indicator-analyte complex, and the binding affinity between indicator and analyte.
