Protein Separation by Electrophoretic–Electroosmotic Focusing on Supported Lipid Bilayers

Abstract

An electrophoretic-electroosmotic focusing (EEF) method was developed and used to separate membrane-bound proteins and charged lipids based on their charge-to-size ratio from an initially homogeneous mixture. EEF uses opposing electrophoretic and electroosmotic forces to focus and separate proteins and lipids into narrow bands on supported lipid bilayers (SLBs). Membrane-associated species were focused into specific positions within the SLB in a highly repeatable fashion. The steady-state focusing positions of the proteins could be predicted and controlled by tuning experimental conditions, such as buffer pH, ionic strength, electric field and temperature. Careful tuning of the variables should enable one to separate mixtures of membrane proteins with only subtle differences. The EEF technique was found to be an effective way to separate protein mixtures with low initial concentrations, and it overcame diffusive peak broadening to allow four bands to be separated simultaneously within a 380 μm wide isolated supported membrane patch.

Lipid-nanostructure hybrids and their applications in nanobiotechnology

“…4,5 These advances have enabled the development of SLB-based membrane-mimicking systems for sensing membrane reactions, studying intercellular signaling and separating membrane species. [6][7][8][9] To simulate and monitor various cell membrane-based processes, the lateral heterogeneity in a subcellular dimension should be reproducibly created and the molecular interaction should be read out with high reproducibility and high sensitivity. Nanomaterials have generated great interest because their size, shape, dimensions and composition-dependent properties are comparable to those of biological molecules and structures, allowing the investigation of biological phenomena at a subcellular level.…”

Functionalized hollow silica nanospheres for His-tagged protein purification

“…The easy separation and purification of proteins, especially lowabundance proteins, are very important in proteomics [1][2][3][4]. Today many purification methods employ a separation step based on specific interactions between immobilized ligands and affinity tags on the protein, and the most common affinity tag is polyhistidine, which can bind to immobilized Ni 2+ or Co 2+ complexes [5][6][7][8].…”

Biofunctionalization of silica microspheres for protein separation

“…To date, various methods such as precipitation, dialysis, ultrafiltration and chromatography are available for purifying target proteins [3,4]. Among them, affinity separation based on the natural biological affinity between biological macromolecules and complementary ligands is of extraordinary significance.…”