Presentation ITC
Presentation
The PEAQ-ITC 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
Manager : Caroline Mas
Office : CIBB room 020
Platform : EMBL room 152
Specific equipment
PEAQ-ITC
PEAQ-ITC 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
• PEAQ-ITC washing module for easy cell and syringe cleaning
Access mode
Users must read the conditions of use of the instruments as well as of the management of the services provided :
 - Access mode
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- General Conditions
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Instrument allocation after user training :- It is mandatory to attend a training session before using this instrument.
- The first two days of use will be done with the assistance of the manager.
- After demonstrating his autonomy, the user will be able to use the instrument in complete independence
- 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 the maintenance fees
Industrials : Please contact us for cost information
Location
The PEAQ-ITC 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 PEAQ_ITC.
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 TRAINING REQUEST : send an email to caroline.mas ibs.fr
PEAQ ITC BookingSamples- 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. 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.
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."
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