Accurate Kd via Transient Incomplete Separation

<div>Current methods for finding the equilibrium dissociation constant, <i>K</i>_d, of protein-small molecule complexes have inherent sources of inaccuracy.</div><div><br></div><div>We introduce “Accurate <i>K</i>_d 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 <i>K</i>_d value. <br></div><div><br></div><div>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 <i>K</i>_d of protein-small molecule complexes.</div>