Isothermal Titration Calorimetry Laboratory
Objective
Isothermal Titration Calorimetry (iTC200 System): iTC200 is used for characterization of molecular interactions of small molecules, proteins, antibodies, nucleic acids, lipids and other biomolecules. Enzyme kinetics, assessment of the effect of molecular structural changes on binding mechanisms, assessment of biological activities is also possible. Direct measure of sub-millimolar to nanomolar binding constants (102 to 109 M-1) and measurement of nanomolar to picomolar binding constants (109 to 1012 M-1) using the competitive binding technique.
Equipment & Details
Isothermal Titration Calorimeter
ITC200 MicroCalorimeter
- Ultrasensitive ITC200 MicroCalorimetry cell with syringe tower
- Control unit for real-time operation and post-run data analysis
- ITC200 washing module for cleaning cell and syringe
- Injection syringes (Hamilton Syringe)
Technical specifications
- Operating Temperature Range: 2 - 80°C
- Response Time: 10 seconds
- Cell Design (volume required): 200μL
- Titration Syringe: 40μL
- Smallest Injection Size: 0.1μL
- Sensitivity range: <0.5 nanocalories (~2 nJ)
Manufacturer details: ITC200 MicroCalorimeter
Malvern Instruments Ltd.
Enigma Business Park,Grovewood Road, Malvern,
Worcestershire, WR14 1XZ, UK
Tel: +44 (0)1684 892456 Fax: +44 (0)1684 892789
system.orders@malvern.com
www.malvern.com
Date of installation: 10th August, 2012
Sample Details
Sample preparation~300 μL of protein sample is required for sample cell and ~40 μL of ligand solution is required for syringe
The buffers in both the sample cell and the titration syringe should be same to avoid the heat of dilution.
Typical starting concentrations are 10-50 μM for the cell and 50-500 μM for the syringe
Syringe concentration ideally 10 x higher than molar concentration of protein in cell, 7 x higher for very tight binding, 15 -20 x higher (or more) for weak binding (Since the syringe volume is ~ 5 fold the cell volume, a 10 fold excess in the syringe will give a final ~ 2 fold excess in the cell)
Precautions:
Flammable liquids should be avoided
Strong acids, detergents and organic solvents should be avoided
Operator should follow proper laboratory procedures in handling and disposing of volatile or hazardous solutions
Assigned Technician:
Bidhan Chandra Sing
Senior Technician/Senior Laboratory Assistantbidhan@adm.iitkgp.ac.in
Isothermal Titration Calorimetry Laboratory
Location: NB / FF / 9, CRF
Facilitator: Prof. Swagata Dasgupta
Location: NB / FF / 9, CRF
Facilitator: Prof. Swagata Dasgupta
Prof. Swagata Dasgupta
Chemistryswagata@chem.iitkgp.ac.in
+91-3222-283306
Utility and Working Principle
Isothermal Titration Calorimetry (ITC) is a well-known analytical technique that can effectively used for investigating the thermodynamic properties of the interaction of macromolecules, small molecules etc. The basic principle of ITC is very simple. It basically measures the heat change (that is either released or absorbed) during a biomolecular binding event. The instrument consists of two cells. One is reference cell which contains water or buffer and other one is sample cell which contains the sample. It is important to keep these both cells at exactly the same temperature. The heat sensing devices detect temperature difference between the cells when binding occurs and give feedback to the heaters, which compensate for this difference and return the cells to equal temperature.