The course of Isothermal Titration Calorimetry data analysis: second part

In the second part of this course, we are going to show you how to perform the analysis of binding isotherms considering an independent sites approach.
This approach uses a reaction scheme based on the binding of the ligand to individual sites present in the receptor and considering that all the sites are independent; thus, it supplies site binding constants.
This approach offers a sole reaction scheme where a receptor with a certain number of sites “n” binds to the ligand.
The sites are grouped into sets to discern between sites that are non-equivalent.

If you want to know more about how to get the stoichiometry (number of sites) and site binding constants with the independent sites approach you can click here:

Stoichiometric and site constants – two approaches to analyze data with AFFINImeter

The first video tutorial presented is about how to use an independent sites approach to perform fittings:


Into another subject, to introduce the second fitting approach that can be performed with AFFINImeter (Stoichiometric equilibria), we will describe how to use the model builder.
This original tool allows to design and apply your own binding model in an easy way. Check the following video to know the versatility of the model builder:

Finally, if you want to try the Model Builder click here:

Model Builder


Stoichiometric and site constants: two approaches to analyze data with AFFINImeter.

The interaction between two species, i.e. a protein and its ligand, is defined by means of the equilibria existing between free and bound species and the binding constant(s) associated to each equilibrium. This scenario can be described in terms or reaction schemes following two approaches:

a) Based on equilibria between existing stoichiometric species, to obtain stoichiometric binding constants and

b) Based on equilibria between the ligand and specific interaction site(s) of the protein, to obtain site binding constants.


The understanding of both approaches/type of binding constants is key for a correct interpretation of the results after data analysis, in order to get key structural and mechanistic information of the binding event; i.e. the presence or absence of cooperative interactions when a ligand binds to a multivalent receptor.
The design of binding models for ITC curve fitting with AFFINImeter can be done following these two approaches, to perform analysis based on stoichiometric and/or site binding constants.

The scientific team of AFFINImeter has just released three NOTES regarding this subject to guide users into the right selection of binding model approach and a better understanding of stoichiometric vs site binding constants.

Comparative table of the two approaches for binding model design available in AFFINImeter
Characteristics of the two approaches for binding model design available in AFFINImeter



Or visit the RESOURCES section of AFFINImeter web page where you find tutorials, webinars, cases of use, among others.