affinity chromatography technology holdup assay MI:2437 A method for high-throughput measurement of protein-ligand binding affinities. A resin is saturated at high concentration with either a control or a bait molecule. The loaded resins are incubated with a prey solution containing either individual purified proteins/domains or complex mixtures. The bait is present in substantial molar excess relative to all potential preys, ensuring non-saturating binding conditions. Each significantly interacting prey is depleted from the solution by the resin, in a proportion related to its own affinity constant for the bait. After incubation, the liquid phases containing the unbound fractions are recovered, without any washing step to maintain thermodynamic equilibrium. For each prey, its amount in the unbound fraction of the bait sample is quantified relatively to its amount in the unbound fraction of the control sample. This relative quantification enables calculation of dissociation constants (Kd), with rigorously validated accuracy and precision. Prey that are well-quantified but not significantly depleted (their affinity for the bait is lower than the assay’s known detection threshold) may be regarded as a negative interaction. 2025-06-17T09:28:11Z PSI-MI jmedina