MULTIPLE-PLUG BINDING ASSAYS USING AFFINITY CAPILLARY ELECTROPHORESIS

This work evaluates the concept of a multiple-plug binding assay to es timate binding constants of proteins to ligands using affinity capilla ry electrophoresis (ACE). This concept is demonstrated using two model systems: carbonic anhydrase B (CAB, EC 4.2.1.1) and vancomycin from S treptomyces orientalis. Multiple plugs of protein, and non-interacting neutral and protein standards, are injected and analysis of the elect rophoretic mobilities of the individual protein plugs, relative to the non-interacting neutral standard, as a function of the concentration of ligand yields values for their binding constants to the protein. Th ese values agree well with those estimated using other assay and ACE t echniques. This technique offers a new and expeditious approach to est imating binding constants of ligands to proteins.