Blow the dust off your old ITC binding experiments, you still can get kinetic information out of them

AFFINImeter

Titration of a

How many experiments have you carried out in your Isothermal Titration Calorimeter during the last years? Do you know that you can reuse them to obtain kinetic information?

 

 

 

KinITC is a methodology recently developed by Philippe Dumas (CNRS, France) to simultaneously get kinetic and thermodynamic information from a standard ITC experiment. The current implementation of kinITC in AFFINImeter is valid only for 1:1 interactions but we intend to extend this for more complex systems in the near future.

 

 

 

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If you have performed experiments of 1:1 interactions in the past, bring them back and use KinITC to get kinetic constants (kon and koff). Under ideal conditions (perfectly clean sample cell, accurate characterization of the response time of your instrument and sufficiently slow kinetics) the resulting values are expected to be equivalent to those obtained by surface plasmon resonance (SPR). The whole process takes just a few seconds and you can analyse multiple files simultaneously.

Blow the dust off your old ITC binding experiments! Go to AFFINImeter!

 

References:

(1) Burnouf D1, Ennifar E, Guedich S, Puffer B, Hoffmann G, Bec G, Disdier F, Baltzinger M, Dumas P. kinITC: a new method for obtaining joint thermodynamic and kinetic data by isothermal titration calorimetry. J. Am. Chem. Soc. 2012 Jan 11; 134(1):559-65. doi: 10.1021/ja209057d. Epub 2011 Dec 16.

 

 

How can I get kinetic information from an ITC experiment?

Eva Muñoz

The elucidation of kinetic aspects of molecular interactions has been gaining interest in many research areas. For instance, the quantitative analysis of binding kinetics helps to a better understanding of the biological function of molecular interactions; it also serves to identify and characterize lead compounds in drugs discovery programs. Getting kinetic information of a binding event requires the use of real-time techniques in which, an observable is monitored as a function of time during the course of the titration.

Isothermal Titration Calorimetry (ITC) has been formally considered a technique to get steady-state binding information. However the primary data of an ITC experiment (ITC raw data), the power vs plot time, is the result of monitoring the heat flow as a function of time. Therefore, the power vs plot time could deliver kinetic information as well.

KinITC (1) is a new analytical tool implemented in AFFINImeter and developed to obtain kinetic information from ITC data of 1:1 interactions. The method consists in determining the Equilibration Time for every peak of the power vs time plot (that is, the time needed to return to baseline after injection) and plot it against the titrant to titrate molar ratio to obtain the so called Equilibration Time Curve (ETC). Noteworthy, a clear sign that the ITC raw data plot contains kinetic information is the increase of the Equilibration Time of the peaks close to mid-titration. Under these conditions, fitting of the ETC yields the dissociation rate constant, koff. Fitting of the corresponding isotherm yields the association constant, KA; ultimately, the association rate constant, kon, is calculated as the product of koff * KA.

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AFFINImeter guest presenter of the Malvern webinar on advanced Isothermal Titration Calorimetry (ITC) data analysis

AFFINImeter
Webinar on advanced Isothermal Titration Calorimetry (ITC) data analysis

The AFFINImeter team are guest presenters of the Malvern webinar on advanced Isothermal Titration Calorimetry (ITC) data analysis. Together we will show how the latest advances in the field of ITC data analysis enable users to “squeeze” the ITC isotherm(s) to get more information than just thermodynamic data and to expand the range of applications of ITC. This webinar will be very helpful for ITC users studying complex interactions, for those who are having difficulties with their data analysis, or simply want to take their analysis to the next level. It is of special interest relevant for researchers from academia and industry working in biomedical applications, particularly in the area of drug design.

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New version with AFFINImeter KinITC Included

AFFINImeter

AFFINImeter is the most complete software for Isothermal Titration Calorimetry data Analysis. You can easily  build your own Binding Models or Perform Global Simultaneous Analysis of several Isotherms, among other features.

In this new AFFINImeter  version we have implemented KinITC, this is a new method to obtain kinetic information from Isothermal Titration Calorimetry Data. With one single titration experiment it calculates the kinetic constants (kon and koff) and the thermodynamic data (KD and ΔH) of 1:1 binding interactions.

AFFINImeter KinITC (soon to be released) offers a general solution for the smart and efficient analysis of the primary signal obtained from ITC experiments. This solution will include an automatic detection of outliers to remove the noise, the detection of the end injection time, the integration of the injection peaks and a preliminary analysis of the peak shape during the titration. The latter feature will allow to provide an estimation of the kinetic constants in the most typical cases where the experiment involves only the formation of 1:1 complexes.

Simulation, advanced ITC data fitting and Kinetics Analysis
Simulation, advanced ITC data fitting and Kinetics Analysis

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Thermodynamic and kinetic aspects of Molecular Recognition Processes

Eva Muñoz

The understanding of molecular recognition processes requires a thorough examination from different perspectives including thermodynamic and kinetic aspects of the binding interaction and structural aspects of the interactants and the complex.

The determination of the binding affinity of two (or more) interactants (i.e. a protein/ligand system), through a steady state analysis provides information on how strong is the complex formed, and it is typically expressed in terms of equilibrium binding constants (association, KA , or dissociation, KD constants). The kinetic analysis of the interaction offers information on how fast the complex is formed and how fast it dissociates, expressed in terms of association and dissociation rate constants, respectively (kon and koff).

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Request an AFFINImeter Online Demo

AFFINImeter

AFFINImeter is a software designed to further exploit the potential of your Isothermal Titration Calorimetry instrument.

Contact us to request an Online Demo

We want to offer our help to guide you through AFFINImeter. If you are interested in the analysis of a particular ITC data and you don’t know which binding model you should apply or how to design it with AFFINImeter, do not hesitate to contact us!

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Meanwhile you can consult our educational material (Videotutorial, Cases of Use, Notes and Webinars) in our Web Page or read the Tutorials and examples section.

Remember that as a launching promotion you can use AFFINImeter free during the first 6 months!

Global fitting analysis of a protein-ligand binding experiment

AFFINImeter

Global fitting analysis of a protein-ligand binding experiment

A few weeks ago AFFINImeter launched an Isothermal Titration data Analysis challenge of the analysis of a protein-ligand binding experiments. The participant had to globally analyze a set of Isothermal Titration Calorimetry experiments using AFFINImeter and get the thermodynamic and structural parameters of the interaction between both molecules (the receptor protein and the ligand).

The participants in this contest had the opportunity to demonstrate their ability to propose the right model for a given binding isotherm as well as to get the corresponding parameters upon fitting using AFFINImeter. On their side, less experienced participants had the opportunity to:

The results recently published by Henlz et al in [Methods 59 (2013) 336-348] were taken as a reference to generate the set of ITC isotherms selected for this contest.

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Complete guide of a global analysis of Isothermal Titration Calorimetry data in AFFINImeter

AFFINImeter

A few days ago AFFINImeter launched an Isothermal Titration Calorimetry (ITC) data analysis challenge. Here the participant had to globally analyse a set of Isothermal Titration Calorimetry experiments using AFFINImeter and get the thermodynamic and structural parameters of the interaction between both molecules (the receptor protein and the ligand).

The participants in this contest had the opportunity to demonstrate their ability to propose the right model for a given binding isotherm as well as to get the corresponding parameters upon fitting using AFFINImeter. On their side, less experienced participants had the opportunity to:

During the last days we though it might be useful to help you throughout the fittings proposed for the contest. Therefore, we have prepared a video that explains, step by step, How to fit CURVE 1. IMPORTANTLY, this information will be of great help to solve step 2, the global fitting of CURVES 1-4.

The data proposed for the contest will remain available here, in case you want to learn more about it or to try it by yourself.

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