Open-tubular capillary electrochromatography of bovine beta-lactoglobulin variants A and B using an aptamer stationary phase

DNA aptamers that form a G-quartet conformation were covalently attached to a capillary surface for open-tubular capillary electrochromatographic sepa ration of bovine beta -lactoglobulin variants A and B, which vary by 2 of t heir 162 amino acid residues. Separation was achieved using a 4-plane, G-qu artet aptamer stationary phase with tris(hydroxymethyl)aminomethane (Tris) or phosphate buffer as the mobile phase. In control experiments, separation did not occur using either an oligonucleotide of similar base composition but which does not form a G-quartet structure, or using capillary zone elec trophoresis on a bare capillary under similar experimental conditions. Sepa ration was achieved using a capillary coated only with the covalent linker molecule. In phosphate buffer, the separations were similar for aptamer-coa ted and linker-only stationary phases, while in Tris buffer, retention time s were almost doubled for the linker-only capillary. When Tris buffer is th e mobile phase, there appears to be weaker interactions between the protein s and the stationary phase that may result in a gentler, less denaturing se paration than is commonly achieved using hydrocarbon-based stationary phase s.