Frontal analysis in microchip capillary electrophoresis: a simple and accurate method for determination of protein-DNA dissociation constant

摘要

Equilibrium constants, such as the dissociation constant (Kd), are a key measurement of noncovalent interactions that are of importance to the proper functioning of molecules in living systems. Frontal analysis (FA) is a simple and accurate capillary electrophoresis (CE) method for the determination of Kd. Microchip CE coupled with laser-induced fluorescence (LIF) detection was used to determine Kd of protein-DNA interactions using the FA method. A model system of immunoglobulin E (IgE) and the IgE-binding aptamer was selected to demonstrate the capability of FA in microchip CE. Because the fluorescence emission was dependent on the dye migration velocity, the velocity of the free aptamer was adjusted to be the same as that of the aptamer-IgE complex by setting up individual separation voltage configurations for the free and bound aptamers. The ratio of the free and bound aptamers in the equilibrium mixture was directly measured from the heights of their plateaus detected at 1.0 cm from the intersection of the microchip, and no internal standard was needed. The Kd of the IgE-aptamer pair was determined as 6 ± 2 nM which is consistent with the reported results (8 nM).