GraphPad Curve Fitting Guide

Key concepts: Competitive binding

Key concepts: Competitive binding

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Key concepts: Competitive binding

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What is competitive binding?

In a competitive binding experiment, you use a single concentration of labeled (hot) ligand and vary the concentration of unlabeled (cold) drugs, and measure binding at equilibrium.

Comparing one- and two-site models

Prism offers models for fitting one or two sites. You can use choices in the Compare tab to compare the two fits. When comparing the fits to the one- and two-site models, use common sense as well as statistics. Don't accept a two site model, if one of the sites is only a tiny fraction of the total, or if its Ki is outside the range of competitor concentrations you used in the experiment.

Ligand depletion

If a large fraction of the added radioligand binds to the receptors, the ligand is depleted so the concentration you added is greater than the free concentration. You need to fit these data to a model that accounts for ligand depletion.

Homologous binding

An homologous binding experiment is one where the labeled and unlabeled ligands have identical affinities for the receptors. Generally this is because the two are chemically identical. Receptor number and affinity are determined by analyzing the competition of varying concentrations of unlabeled ligand for one (or better, two) concentrations of labeled ligand. Prism offers a special model for fitting homologous competition experiments.

Allosteric modulators

Allosteric modulators can alter radioligand binding, even though they bind to different sites. Since the hot and cold ligands bind to different sites, the term 'competition' is not apt, but we include this model here because the experimental design is the same as used for competitive binding. Prism can fit binding inhibition (or augmentation) by an allosteric modulator based on the ternary complex model. Note that this model assumes the allosteric modulator is present in excess, so is not depleted by binding to the receptors.