MicroCal VP-ITC

Presentation

The MicroCal VP-ITC is a highly sensitive 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.

https://www.malvernpanalytical.com/fr/products/technology/microcalorimetry/isothermal-titration-calorimetry/

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

Manager : Caroline Mas
Office : CIBB room 017
Instrument : CIBB room 001

Specific equipment

VP-ITC

VP-ITC

VP-ITC Features :
• Active sample cell volume 1.4mL ( 2mL total for filling sample cell)
• Active syringe volume 300 µL ( 500 µL total for filling the syringe)
• Non-reactive Hastelloy® cells for excellent chemical resistance
• Precision liquid delivery system for accurate and reproducible injections
• User-selectable mixing speeds to match sample conditions
• Operating temperature range of room temperature to 50°C
• Peltier controlled for rapid temperature equilibration

Access mode

Users must read the conditions of use of the instruments as well as of the management of the services provided :

• 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. It is mandatory to attend the 1/2 day training session before using this instrument - When needed, please ask to the platform manager
• The platform is accessible to local PSB researchers, external academics (Instruct) and industrials
• The platform manager will provide assistance for :
  • _ experimental design
  • _ data collection
  • _ samples considerations
  • _ data analysis
• Contact the platform manage to arrange a meeting if necessary

• 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

Cost

Academics : Participation to themaintenance fees
Industrials : Please contact us for cost information

Location

The VP-ITC is located in the CIBB building, room 001

How to make a request

A specific formation is mandatory before the use of the instrument VP-ITC.
Experienced users typically perform about 1-3 experiments per day.

To make a booking request, fill this REQUEST FORM HERE

VP-ITC Booking

Biophisics Platform Booking Calendar

Samples

• An example of a preliminary experiment is to inject aliquots of the titrant (300 µL at concentration 10 times higher than the protein, 100 µM-1mM) into the sample cell containing the protein solution (1.4mL, at 10-100 µM)

• To estimate the concentration to use in the “sample cell”, you can calculate the C value. This unit-less parameter is the critical parameter which determines the shape of the binding isotherm.
  It is given by this equation :
  

  C=[M]/Kd*n

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. Avoid β mercaptoethanol, prefer to use 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.

• Only non-pathogenic biological samples are accepted. Decree of the July 18, 1994 establishing the list of biological pathogens, amended by Decrees of April 17, 1997 and June 30, 1998 (Decrees in French). The list of biological pathogens is available on the website of the IPBS.

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 UAR platforms :
"This work used the platforms of the Grenoble Instruct-ERIC center (ISBG ; UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-0005-02) and GRAL, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003). We thank Caroline Mas for assistance and/or access to the biophysics platform."