Abstract
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.
Highlights
Quantitative microdialysis is a traditional, yet powerful technique for the analysis of two important properties of small molecules of interest [1,2,3,4]: (a) the diffusability of small molecules, which is affected by their intramolecular aggregation and their surface binding properties, and (b) the determination of their affinities to target proteins, using equipment found in many laboratories
To determine a KD based on a pt derived from single point measurements open the Python file entitled ‘03_deriveKD_from_pt.py’ in a text or code editor and assign the following variables, taking care to input the correct value for pt as the ratio of compound concentration the the red versus white chambers and run the program to read out the KD: a. l0 = 50, Compound concentration achieved after equilibrium in both chambers in the absence of the protein
Since KD determination is achieved by the accurate comparison of compound concentrations in the two chambers, or by inferring either one from the other in the case of only using one concentration, it is important that the experimental setup is such that a sensitive quantitative and accurate concentration determination is possible over the range of conditions of most interest
Summary
Quantitative microdialysis (qμD) is a traditional, yet powerful technique for the analysis of two important properties of small molecules of interest [1,2,3,4]: (a) the diffusability of small molecules, which is affected by their intramolecular aggregation and their surface binding properties, and (b) the determination of their affinities to target proteins, using equipment found in many laboratories. Dissolved test compounds may freely move between two chambers partitioned by a semipermeable membrane that allows only small molecules to pass through. Along with these freely moving compounds, one chamber contains either the target protein (to which it is assumed or known to bind to) or one or more control proteins (which serve to test the selectivity of the target protein binding reaction). A thermodynamic equilibrium reached in the absence of a protein after a certain incubation time should result in equal concentrations of free compound in the two chambers. If the compound engages in nonspecific binding towards the material of the chambers or the separating membrane, different compound concentrations will be detected between the two chambers, using any
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
