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.
Keywords: KD determination; affinity determination; aggregation; chromatography; dialysis; drug discovery; label free screening; nonspecific binding; promiscuity; protein binding analysis.