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Accurate Kd via Transient Incomplete Separation

preprint
revised on 22.07.2019 and posted on 23.07.2019 by Nicolas Sisavath, Jean Luc Rukundo, J.C. Yves Le Blanc, Victor A. Galievsky, Jiayin Bao, Sven Kochmann, Alexander S. Stasheuski, Sergey N. Krylov
Current methods for finding the equilibrium dissociation constant, Kd, of protein-small molecule complexes have inherent sources of inaccuracy.

We introduce “Accurate Kd via Transient Incomplete Separation” (AKTIS), an approach that is free of known sources of inaccuracy. Conceptually, in AKTIS, a short plug of the pre-equilibrated protein-small molecule mixture is pressure-propagated in a capillary, causing transient incomplete separation of the complex from the unbound small molecule. A superposition of signals from these two components is measured near the capillary exit as a function of time, for different concentrations of the protein and a constant concentration of the small molecule. Finally, a classical binding isotherm is built and used to find accurate Kd value.

Here we prove AKTIS validity theoretically and by computer simulation, present a fluidic system satisfying AKTIS requirements, and demonstrate practical application of AKTIS to finding Kd of protein-small molecule complexes.

Funding

Natural Sciences and Engineering Research Council of Canada (NSERC) STPG-P 521331-2018

Mitacs Canada IT08656

History

Email Address of Submitting Author

skochman@yorku.ca

Institution

York University, Toronto

Country

Canada

ORCID For Submitting Author

0000-0001-7423-4609

Declaration of Conflict of Interest

no conflict of interest

Version Notes

This version now includes a thumbnail with the specs given by Marshall Brennan (https://twitter.com/indianalytics/status/1116762338772828161).

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