Tag: Isotherm fitting

ITC data analysis contest – New submission Date

AFFINImeter

A few days ago AFFINImeter launched an Isothermal Titration Calorimetry (ITC) data analysis contest. At this point no one has been able to finish the challenge, eventhough there is a considerable amount of participants. Since the main goal of the contest is to give all our AFFINImeter users an educational tool to get experience in the process of ITC data analysis.

Therefore, we would not be satisfied if some of you ran  out of time. For this, we have decided to extend the contest deadline to January 31st

Join the Challenge

We encourage all people interested in molecular binding analysis, not only based on ITC data but also on any other experimentaltechnique, to participate in this challenge. If you are an expert in the analysis of thermodynamic data this is a nice opportunity for you!

Find the best combination of model & parameters for the target data and win a Kindle Fire HD 7 just sending us your results.

tablet kindle fire HD7
Isothermal Titration Calorimetry Data Analysis Contest

If you are a beginner in the analysis of ITC or, in general, of binding experiments, this challenge is also perfect for you since it will give you the opportunity to learn how to analyse actual ITC measurements. Our scientific team has selected some really interesting experiments and prepared some instructions that will guide you in the analysis process.

Learn the difference between stoichiometric and site equilibrium constants

By trying to solve this contest you will learn the difference between stoichiometric and site equilibrium constants, you will see how to easily perform a global analysis of several isotherms, and even how to find the, eventually unknown, concentration of active protein in a given experiment. Sign up HERE  and you will get free access to AFFINImeter. Please, do not hesitate to encourage your labmates to join us. People from the whole world are already participating!

Download Poster here

How to perform a Global Fitting Analysis?

AFFINImeter

The Global fitting of multiple isotherms is one of the advanced tools that AFFINImeter offers to facilitate the analysis and interpretation of isothermal titration experiments and to expand the range of applications of this technique.

The following video tutorial describes the global fitting of three isotherms of a displacement assay describing, a receptor interacting with a tight ligand, with a weak ligand, or with both ligands simultaneously, in a competitive experiment where the ligands are mixed in the syringe of the ITC equipment.

 

If you want to know more about global fittings with AFFINImeter you can also download the case of use “Global Analysis in ITC Displacement Titrations with AFFINImeter” that describes a Displacement Titration Assay to determine the thermodynamics of HIV-protease with indinavir, a high-affinity binder, and with acetyl-pepstatin, a weaker ligand.

ITC displacement titrations offer an attractive alternative to standard assays when working with ultra-high or ultra-low- affinity interacting systems. The method requires the fitting of at least two isotherms that share various adjustable parameters. The case study exemplifies the potential advantages of using AFFINImeter in ITC displacement assays. The software offers unique advanced tools that enhance the robustness of the method and makes it more versatile, facilitating the acquisition of reliable thermodynamic data from ultra-high of ultra-low affinity systems. Thus, it opens a door for new applications of the displacement assay.

 

 

 

Competitive Binding Assay

AFFINImeter

Competition assays

Competitive binding assays where two (or more) ligands bind to the same receptor have become common experiments in many research areas, from basic investigations to innovation in the pharmaceutical industry. These assays can be done in different formats, i.e. through a displacement assay where ligand “L1” is displaced by ligand “L2” from a preformed complex “L1-receptor” or via titration of a receptor solution with a mixture of L1 +L2. Either way, the competitive binding assay provides rich thermodynamic and structural information of the various binding events taking place during the course of the experiment. Thus, Isothermal Titration Calorimetry (ITC) competition assays performed in a displacement format have been revealed as an efficient tool for the quantitative analysis of very high- / low- interactions, with application in the field of fragment based drug screening (ref).

Analysis of competition experiments with AFFINImeter

The versatility of the experimental setup in AFFINImeter-ITC permits the analysis of ITC competition experiments in its various formats. As an illustration, the following lines describe the analysis of an ITC isotherm resulting from a competitive experiment where a solution of a receptor in titrated with mixture of two competing ligands.

AFFINImeter contains a series of examples with which users can practice and learn the overall process of data fitting: from equipment and data uploading to fitting model design and data fitting.

In this post we will review an example of a competitive model fitting model used to analyze the experiment data of two ligands in the syringe competing for binding to the same receptor.

Practical case: competitive binding model

The AFFINImeter example “competitive binding model” illustrates an ITC experiment where two ligands, “A” and “B” compete with each other for binding to the receptor “M”.

Drawing of a competitive binding model
Competitive Binding model scheme

This situation corresponds to a binding model consisting of three free species (A, B and M) two binding equilibria representing the interaction of M with A and M with B

The model was designed with the “reaction builder” and stored in “models”.The equipment used is decribed and stored in “equipments”. The dataseries is uploaded and stored in “dataseries”. When the dataseries is uploaded, the user has to complete the information relative to the equipment used and the species concentration. In this particular case (where there is a competitor “B”).

How to fit a project with AFFINImeter

 

1- Go to PROJECT MANAGEMENT and create a new PROJECT an a new FIT SUBPROJECT.

  • Add the dataseries created previously to the subproject.
  • Select the model created previously.
  • Keep all the default values in FitSetting.

2- Press Run button.

This Steps are described in the following Video Tutorial:

You can follow this tutorial in AFFINImeter, the Experimental Data and Binding model are stored in your own AFFINImeter account. If you hasn’t registered yet go to the AFFINImeter Software WebPage to get your account.

 

 

Ref: W. B. Turnbull, Divided we fall? Studying low-affinity fragments of ligands by ITC. GE Healthcare Life Sciences protocol, 2011, pp 1-11.