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.
InstitutionYork University, Toronto
ORCID For Submitting Author0000-0001-7423-4609
Declaration of Conflict of Interestno conflict of interest
Version NotesThis version now includes a thumbnail with the specs given by Marshall Brennan (https://twitter.com/indianalytics/status/1116762338772828161).