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3 ©2018 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. binding affinity, the dissociation constant provides a measurement of how much ligand-protein complexes are formed at equilibrium. In binding kinetics, the k off and k on rates describe, respectively, the rate at which molecules dissociate and associate. These reaction rates may be interesting parameters to researchers who want to evaluate time-dependent changes in ligand-protein engagement. Binding kinetics vs. binding affinity in basic research Measuring binding affinity has many applications including monitoring the regulation of cellular pathways, screening compounds and drug candidates, and testing structure-function relationships. Kinetic analysis provides a level of information that is important for a few specialized areas of research and late-stage drug development. It might also be helpful to know the binding kinetics of protein-ligand complexes during nuclear magnetic resonance spectroscopy experiments. Since interactions with the same K d can have different kinetics, the rate constants k off / k on are helpful for determining binding modes. Because of this, the U.S. FDA requires binding kinetics information for approving drugs in cases when binding or immunochemical properties are part of the activity attributed to the protein product 1 . However, for most basic research and early-stage drug development, knowing binding kinetics is not always necessary. In order to arrive at sufficient Two approaches to measuring ligand-protein interaction There are two approaches for measuring the interaction between a protein and another molecule (ligand). One is by measuring binding affinity, which refers to the strength of the interaction between the two molecules. The other one is by looking at binding kinetics, which focuses on the rate of interaction. The dissociation constant (K d ) is a biophysical constant describing binding affinity between two molecules. The K d corresponds to the concentration of ligand necessary to have 50% of the receptor in the bound state under certain conditions. So, the K d will tell you if a complex is formed at given ligand and receptor concentrations. K d is a parameter that lets researchers rank the strengths of the interactions that occur between molecules. The smaller the dissociation constant the more tightly bound the ligand is, thus the higher the affinity is between the two molecules. This information is valuable for research because it allows for the quantification of a protein molecule's reaction with a ligand and predicts the interaction behavior under physiological conditions. The K d is used to calculate the ratio of complex to unbound molecules at given concentrations. On the other hand, binding kinetics parameters describe how long it takes until equilibrium is reached for interaction between the two molecules by analyzing the speed at which the interactions occur. In

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