1. Transient Incomplete Separation of Species with Close Diffusivity to Study the Stability of Affinity Complexes.
- Author
-
Wang TY, Rukundo JL, Le ATH, Ivanov NA, Le Blanc JCY, Gorin BI, and Krylov SN
- Subjects
- Computer Simulation, Protein Binding, Entropy, Electrophoresis, Capillary methods, Oligonucleotides chemistry
- Abstract
Large molecules can be generically separated from small ones, though partially and temporarily, in a pressure-driven flow inside a capillary. This transient incomplete separation has been only applied to species with diffusion coefficients different by at least an order of magnitude. Here, we demonstrate, for the first time, the analytical utility of transient incomplete separation for species with close diffusion coefficients. First, we prove in silico that even a small difference in diffusivity can lead to detectable transient incomplete separation of species. Second, we use computer simulation to prove that such a separation can be used for the reliable determination of equilibrium dissociation constant ( K
d ) of complexes composed of similar-sized molecules. Finally, we demonstrate experimentally the use of this separation for the accurate determination of Kd value for a protein-aptamer complex. We conclude that "accurate constant via transient incomplete separation" (ACTIS) can serve as a reference method for affinity characterization of protein-aptamer binding in solution.- Published
- 2022
- Full Text
- View/download PDF