Quantitative microdialysis: Experimental protocol and software for small molecule protein affinity determination and for exclusion of compounds with poor physicochemical properties

Quantitative microdialysis is a traditional biophysical affinity determination technique.
In the development of the detailed experimental protocol presented, we used commercially available
equipment, rapid equilibrium dialysis (RED) devices (ThermoFisher Scientific), which means that it is
open to most laboratories. The target protein and test compound are incubated in a chamber partitioned
to allow only small molecules to transition to a larger reservoir chamber, then reversed-phase high
performance liquid chromatography (RP-HPLC) or liquid chromatography–mass spectrometry
(LC–MS) is used to determine the abundance of compound in each chamber. A higher compound
concentration measured in the chamber that contains the target protein indicates binding. As a novel,
and differentiating contribution, we present a protocol for mathematical analysis of experimental data.
We provide the equations and the software to yield dissociation constants for the test compound-target
protein complex up to 0.5 mM KD, and we quantitatively discuss the limitations of affinities in relation
to measured compound concentrations.