Two Original Experimental Setups for Staircase Frontal Affinity Chromatography at the Miniaturized Scale
Résumé
Frontal affinity chromatography is a powerful, underappreciated technique for the qualitative (screening) and quantitative (K-d determination) evaluation of biological interactions. Its development has been previously hampered by its sample consumption, limited throughput, and lack of dedicated instrumentation especially at a miniaturized scale. This work describes two original experimental devices allowing nano-frontal affinity chromatography titrations (nano-FAC) to be automatically implemented in the time-saving staircase mode. The first nanoFAC system utilizes a capillary electrophoresis device (7100 CE Agilent system) in the pressurization mode with in situ UV detection. The second nano-FAC experimental setup implements a nano-LC device (Ultimate 3000 Thermo) modified with a 10-port valve equipped with two superloops (loop volume, 5 mu L) operating alternatively and automatically in a single run. The benefits and drawbacks of each approach are exemplified using two model protein-ligand interactions (concanavalin A-mannose and concanavalin A-glucose). The two methods result in concordant dissociation constants (K-d) and number of active site (B-act) values, obtained in a fully automated manner, with low sample consumption and good throughput.