Extending Affinity Capillary Electrophoresis to Intracellular Membrane Targets

Affinity capillary electrophoresis (ACE) is a powerful technique for quantifying biomolecular interactions. Despite its demonstrated utility for soluble proteins, its application to membrane targets has remained largely unexplored because these proteins strongly depend on their native lipid environment for structural and functional integrity.
In this work, we present a strategy that extends partial-filling ACE to intracellular membrane proteins maintained in their native cellular context. Golgi microsomes isolated from Caki-1 renal carcinoma cells were used as a source of native membrane vesicles containing UDP-glucose ceramide glucosyltransferase (UGCG), a Golgi-resident enzyme implicated in cancer progression, drug resistance, and immune regulation. Nanoparticle tracking analysis confirmed the presence of fairly monodisperse vesicles (~150 nm), while activity assays demonstrated preservation of UGCG function.
A dedicated data analysis framework was implemented to extract binding parameters under low target abundance conditions and to account for nonspecific interactions, overcoming limitations traditionally associated with ACE studies. The methodology supports both direct and competitive formats, enabling characterization of ligands with diverse physicochemical properties.
These results demonstrate that ACE can be successfully adapted to investigate ligand recognition processes occurring at the surface of native intracellular membrane compartments. Beyond UGCG, this platform opens new opportunities for studying therapeutically relevant membrane-associated targets in complex biological environments, including intracellular vesicles, extracellular vesicles, and other membrane-bound nanoparticles.