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Using Nonequilibrium Capillary Electrophoresis of Equilibrium Mixtures (NECEEM) for Simultaneous Determination of Concentration and Equilibrium Constant.
- Source :
-
Analytical Chemistry . 3/3/2015, Vol. 87 Issue 5, p3099-3106. 8p. - Publication Year :
- 2015
-
Abstract
- Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) is a versatile tool for studying affinity binding. Here we describe a NECEEM-based approach for simultaneous determination of both the equilibrium constant, Kd, and the unknown concentration of a binder that we call a target, T. In essence, NECEEM is used to measure the unbound equilibrium fraction, R, for the binder with a known concentration that we call a ligand, L. The first set of experiments is performed at varying concentrations of T, prepared by serial dilution of the stock solution, but at a constant concentration of L, which is as low as its reliable quantitation allows. The value of R is plotted as a function of the dilution coefficient, and dilution corresponding to R = 0.5 is determined. This dilution of T is used in the second set of experiments in which the concentration of T is fixed but the concentration of L is varied. The experimental dependence of R on the concentration of L is fitted with a function describing their theoretical dependence. Both Kd and the concentration of T are used as fitting parameters, and their sought values are determined as the ones that generate the best fit. We have fully validated this approach in silico by using computer-simulated NECEEM electropherograms and then applied it to experimental determination of the unknown concentration of MutS protein and Kd of its interactions with a DNA aptamer. The general approach described here is applicable not only to NECEEM but also to any other method that can determine a fraction of unbound molecules at equilibrium. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 00032700
- Volume :
- 87
- Issue :
- 5
- Database :
- Academic Search Index
- Journal :
- Analytical Chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 101827618
- Full Text :
- https://doi.org/10.1021/acs.analchem.5b00171