Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety - Facilities & Instrumentation

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Facilities & Instrumentation - Biomolecular Physics Laboratory

Isothermal Titration Calorimetry (ITC)

Isothermal Titration Calorimetry (ITC) is the gold standard for measuring biomolecular interactions. ITC simultaneously determines all binding parameters (n, K, ΔH and ΔS) in a single experiment – information that cannot be obtained from any other method. When substances bind, heat is either generated or absorbed. ITC is a thermodynamic technique that directly measures the heat released or absorbed during a biomolecular binding event.

Measurement of this heat allows accurate determination of binding constants (Kb), reaction stoichiometry (n), enthalpy (ΔH) and entropy (ΔS), thereby providing a complete thermodynamic profile of the molecular interaction in a single experiment. Because ITC goes beyond binding affinities and can elucidate the mechanism of the molecular interaction, it has become the method of choice for characterizing biomolecular interactions. Our lab is equipped with a MCS-ITC micro-calorimetry system, which allows ultrasensitive characterization of the thermodynamics of a reaction in solution.

Applications

The MCS-ITC can be used:

To study any type of reactions including antigen-antibody, DNA-drug, receptor-target, protein-ligand, and protein-protein, among others.
To determine reaction stoichiometry.
To measure binding constants.
To obtain thermodynamic properties of binding such as enthalpy, entropy and Gibbs free energy.

Specifications

Normal operating temperature: 0°C – 80°C
Sample (macromolecules) cell and reference cell volumes: ~ 1.4 mL
Typical macromolecule concentrations required (depending on magnitude of binding constant): 20 mM – 1 mM.
Titration syringe (ligand): total volume of 50 mL, 100 mL and 250 mL available; injection volumes typically 5-10mL.

Typical ligand concentrations required: 10- 50 times the macromolecule concentration, depending on syringe size used.

Differential Scanning Calorimetry (DSC)

Differential Scanning Calorimetry (DSC) is unsurpassed for understanding the stability of biological systems. DSC directly measures heat changes that occur in biomolecules during controlled increase or decrease in temperature, making it possible to study materials in their native state. DSC measures the enthalpy (∆H) of unfolding due to heat denaturation. A biomolecule in solution is in equilibrium between the native (folded) conformation and its denatured (unfolded) state. The higher the thermal transition midpoint (Tm), when 50% of the biomolecules are unfolded, the more stable the molecule. DSC is also used to determine the change in heat capacity (ΔCp) of denaturation.

Our lab is equipped with the VP-DSC differential scanning calorimeter which allows the analysis of dilute macromolecular solutions.

Applications include:

Protein stability and folding.
Liquid biopharmaceutical formulations.
Process development.
Protein engineering.
Antibody domain studies.
Characterization of membranes, lipids, nucleic acids and micellar systems.
Assessment of the effects of structural change on a molecule’s stability.
Measurement of ultra-tight molecular interactions (up to 10²⁰ M^-1).
Assessment of biocomparability during manufacturing.

VP-DSC features:

Active cell volume ~ 0.5 ml.
Non-reactive Tantalum 61™ cells for excellent chemical resistance.
Fixed-in-place cells for reproducible ultrasensitive performance with low maintenance.
Operating temperature range of –10ºC to +130ºC.
Peltier element for precise temperature control.
User selectable temperature scan rates (0ºC to 90ºC per hour upscans) and range for application versatility.
Unparalleled sensitivity and reproducibility.
Three user selectable response times for maximum performance.
Allows studies of fast or slow transition processes.

Self-contained pressurizing system (0-45 psi) for studying solutions above their boiling point.

Fluorescence spectroscopy

Fluorescence is a spectrochemical method of analysis where the molecules of the analyte are excited by irradiation at a certain wavelength and emit radiation of a different wavelength. The emission spectrum provides information for both qualitative and quantitative analysis.

We are equipped with a highly sensitive QuantaMaster™ 40 fluorescence spectrometer.

Applications

Specifications

Detection Limit: 460 attomolar fluorescein in 0.1 M NaOH
Signal to Noise Ratio: 10,000:1 or better (350 nm excitation, 5 nm spectral bandpass, 1 s integration time)
Data Acquisition Rate: 50,000 points/sec. to 1 point/100 sec
Emission Range: 180 nm to 680 nm
Light Source: High efficiency continuous Xenon arc lamp
Resolution: 0.25 nm
Detection: Photon counting/analog

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