Version :
Integrated Structural Biology Grenoble

Contact person(s) related to this article / Caroline Mas

Presentation ITC

Presentation

The MicroCal iTC200 is a highly sensitive, low volume isothermal titration calorimeter for the label-free in solution study of biomolecular interactions. It delivers direct measurement of all binding parameters in a single experiment. Requiring no modification of binding partners, either with fluorescent tags or through immobilization, ITC measures the affinity of binding partners in their native states. It works by directly measuring the heat that is either released or absorbed during a biomolecular binding event.

By measuring heat transfer during binding, ITC allows the direct determination of binding stoichiometry (n), of binding constants (KD), and variations of enthalpy (ΔH) and entropy (ΔS). Analyses at several temperatures give access to the variation of heat capacity (ΔCp) linked to the formation of a molecular complex. This provides a complete thermodynamic profile of the molecular interaction.
The enthalpy (ΔH) and the entropy (ΔS) contain information about the type of interaction and reflect the nature of the forces driving the binding.

http://www.malvern.com/en/products/technology/isothermal-titration-calorimetry/default.aspx

Applications

  • Characterization of molecular interactions established by:
    • Protein-small molecule ( lipids, sugars, peptides, compounds,...)
    • Enzyme-inhibitor
    • Antibody-antigen
    • Protein-protein
    • Protein-DNA
    • Protein-Metal ion-
  • Assessment of biological activity.
  • Enzyme kinetics

Key Words

Molecular interaction, Dissociation constant, Enthalpy, Entropy, Stoichiometry

Staff

Platform engineer: Caroline Mas
Office: CIBB room 020
Platform: EMBL room 152

Specific equipment

iTC200

iTC200 Features:
• Active sample cell volume 200 µL (350 µL total for filling sample cell)
• Active syringe volume 40 µL (70 µL total for filling the syringe)
• Non-reactive Hastelloy® cells for excellent chemical resistance
• Fixed-in-place cells for reproducible ultrasensitive performance with low maintenance
• Precision liquid delivery system for accurate and reproducible injections (typically 1 tp 3 µL)
• User-selectable mixing speeds to match sample conditions
• Operating temperature range of 2°C to 80°C
• Peltier controlled for rapid temperature equilibration
• iTC200 washing module for easy cell and syringe cleaning

ITC Data Analysis with Origin
Malvern iTC200 Manual

Access mode

  • The platform is accessible to local PSB researchers
  • Service: routine experiments performed for punctual users
  • Instrument allocation after user training: for users who have completed training to gain operational autonomy time is allocated to them to perform and analyze their experiments
  • Scientific collaboration: projects in which the Biophysical platform is strongly involved. In this case, experiments are designed, performed and analyzed in tandem between the scientific responsible and the applicant

Location

The iTC200 is located at the EMBL, laboratory 152, 1st Floor (Map Here)

How to make a request

A specific formation is mandatory before the use of the instrument iTC200.
Experienced users typically perform about seven experiments per day, so we offers booking for one day as starting.
Booking: 2 days maximum

To make a booking request, fill this REQUEST FORM HERE

ITC200 Booking

Samples

  • An example of a preliminary experiment is to inject aliquots of the titrant (40 µL at concentration 10 times higher than the protein, 100 µM-1mM) into the sample cell containing the protein solution (200 µL, at 10-100 µM)
  • To estimate the concentration to use in the “sample cell”, you can calculate the C value. This unitless parameter is the critical parameter which determines the shape of the binding isotherm.
    It is given by this equation:

    C=[M]*Kd*n

With:

[M]: total macromolecule concentration in the cell at the start of the experiment

Kd: binding constant

N: stoichiometry

Very good if 10< C <100

Good if 5< C <500

Ok if 1-5 and 500-1000

Not good if C<1 and C>1000

  • An ITC experiment consists of two titrations:
    • titrant (syringe) injected to the macromolecule (sample cell)
    • titrant (syringe) injected to the buffer (sample cell)
  • No buffer or salt limitations, except DTT as reducing agent and unstable chemicals. Use β mercaptoethanol or TCEP ( the TCEP is not stable in Phosphate Buffer)
  • Importantly, the buffer composition must be identical for the titrant and cell solutions
  • The technique does not require any labelling or modification of materials. ITC accuracy is not affected by ligand and protein sizes

Follow up / acknowledgements

The user agrees to promote the obtained results by mentioning the platform in the acknowledgements in case of publication or scientific communication and to communicate the reference of the article to the responsible for the platform.

Acknowledgements in publications:

Please find below the sentence that has to be written in all publications based on our UMS platforms:
"This work used the platforms of the Grenoble Instruct-ERIC Center (ISBG : UMS 3518 CNRS-CEA-UGA-EMBL) with support from FRISBI (ANR-10-INBS-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). We thank Caroline Mas and/or Marc Jamin, for assistance and/or access to the Biophysical platform."