Frontal analysis in microchip CE: a simple and accurate method for determination of protein-DNA dissociation constant.
Equilibrium constants, such as the dissociation constant (K(d)), are a key measurement of noncovalent interactions that are of importance for the proper functioning of molecules in living systems. Frontal analysis (FA) is a simple and accurate CE method for the determination of K(d). Microchip CE coupled with LIF detection was used to determine K(d) of protein-DNA interactions using the FA method. A model system of 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 K(d) of the IgE-aptamer pair was determined as 6 +/- 2 nM which is consistent with the reported results (8 nM).
Gong
Maojun
M
Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221-0172, USA.
Wehmeyer
Kenneth R
KR
Limbach
Patrick A
PA
Heineman
William R
WR
D052157:Aptamers, Nucleotide / Q000032:analysis / Q000737:chemistry*; D019075:Electrophoresis, Capillary; D046209:Electrophoresis, Microchip / Q000379:methods*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007073:Immunoglobulin E / Q000032:analysis / Q000737:chemistry*
C519608:6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein; D052157:Aptamers, Nucleotide; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007073:Immunoglobulin E