Two Original Experimental Setups for Staircase Frontal Affinity Chromatography at the Miniaturized Scale

Abstract

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 nano-FAC 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 μL) 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.

Preparation of miniaturized hydrophilic affinity monoliths: Towards a reduction of non-specific interactions and an increased target protein density

“…The detection was achieved in-situ on an empty section of the column, right after the end of the monolith, thanks to a diode array detector operated in multi-wavelength mode. [18] The revelation window on the capillary was obtained by burning away the external coating of the capillary. The analyses were all carried out under controlled room temperature at 25°C.…”

“…These experiments highlight the benefits associated with reduced non-specific interactions and an increased target protein density. All these experiments are carried out by frontal affinity experiments (with a dedicated in-house developed instrumentation [18]) as described in the Material and Methods Section.…”

Optimization of the Preparation of Hydrophilic Poly(DHPMA-co-MBA) Monolithic Capillary Columns: A New Support for Affinity Chromatography

“…where F is the flow rate to the dead time, V 0 the dead volume of the column and k ns the retention factor of the solute due to non-specific interactions. To determine the number of streptavidin active binding sites that were available after grafting, HABA (K d = 100 µM) was percolated as a test solute with increasing concentrations (5, 10, 50, 100 and 200 µM solutions were prepared in phosphate buffer, 67 mM, pH = 7.4) without a rinsing step between the percolations of the different concentrations (staircase experiments) [23]. The number of ligands captured in each step was determined, and the cumulative number of ligands captured was calculated by summing the number captured in all steps.…”

Optimization of the Preparation of Hydrophilic Poly(DHPMA-co-MBA) Monolithic Capillary Columns: A New Support for Affinity Chromatography

“…where F is the flow rate to the dead time, V0 the dead volume of the column and kns the retention factor of the solute due to non-specific interactions. To determine the number of streptavidin active binding sites that were available after grafting, HABA (Kd = 100 μM) was percolated as a test solute with increasing concentrations (5, 10, 50, 100 and 200 μM solutions were prepared in phosphate buffer, 67 mM, pH = 7.4) without a rinsing step between the percolations of the different concentrations (staircase experiments) [23]. The number of ligands captured in each step was determined, and the cumulative number of ligands captured was calculated by summing the number captured in all steps.…”