Extensions of the kinetics of competitive binding equation for rapid competitor kinetics or alternative order of reagant addition.

The analysis of the kinetics of competitive binding, (Motulsky and Mahan, 1984;  built-in to Prism) has been extended in two ways:

  • To work with different orders of addition of receptor, hot compound and cold compound. The kinetics of competitive binding equation was designed for the most commonly-used assay format, in which receptor is exposed to both ligands simultaneously, i.e. receptor is added last. There are occasions when the ordering of reagent addition is different, for example preincubation of receptors with unlabeled compound, or with labeled ligand

  • To work with rapidly-dissociating compounds. The kinetics of competitive binding equation was developed for unlabeled ligands that dissociate sufficiently slowly that the fit provides reasonable estimates of the kinetic parameters. When the unlabeled ligand dissociates rapidly, an ambiguous fit often results because the ligand equilibrates so rapidly that the kinetic assumption no longer applies.In this scenario, the data can be better fit by an equation that assumes the unlabeled ligand rapidly equilibrates with the free receptors. 

Sam Hoare of Pharmechanics has created a Prism file and PowerPoint presentations to learn about and use these equations:

  • Download a Prism file with sample data and the new equations.
  • This Powerpoint presents the background and explains how to use the equation for rapidly dissociating cold ligand (use Slideshow view).
  • This Powerpoint presents the background, and details for analyzing the kinetics of competitive binding with a different order of addition of reagents (use Slideshow view).
  • This Powerpoint file and Prism file provide additional information and resources (new nonlinear regression model and step-by-step instructions for its application) for a simplified washout protocol frequently used for kinase enzyme inhibitors.


  • These equations are not supported by GraphPad Software. Please email Pharmechanics with any questions or suggestions.
  • Once the template has been opened and saved for the first time, the new equations will be appended to the list of user-defined equations, so there is no need to reopen the template again to use the equations. 


The kinetics of competitive binding equation was described by Motulsky and Mahan in:

Molecular Pharmacology 25:1-9, 1984


The equations for the alternative assay formats are described in the following references


J Recept Signal Transduct Res 29:84-93, 2009

J Biomol Screen 21:729-737, 2016

J Pharmacol Toxicol Methods 89:26-38, 2018



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