Isothermal Calorimetry Flashcards
What are the advantages of ITC?
Provide a full thermodynamic description: KD, n, delta H and delta S
No labelling required
Low affinity interactions can be measured.
What are the disadvantages of ITC?
Expensive equipment required, relatively high concentrations of sample required.
Describe the steps of ITC?
Load sample and control cell, set temperature of micro calorimeter and ensure it is met, load a syringe with ligand and place over sample cell, inject small aliquots of ligand into the sample cell until the ligand is in excess and no further temperature changes are recorded.
Describe how results are measured and recorded?
The reaction dependent upon being Exothermic or endothermic will produce upwards or downwards peaks on a graph compared to the set temperature. The quantity of heat measured is indirectly proportional to the amount of binding. Area of each peak is integrated and plotted against the molar ratio of ligand:protein.
How can ITC be used in drug design?
Quantifying binding activity, candidate selection and optimisation, measurement of thermodynamics and active concentration, characterisation of mechanism of action, conformation of intended binding targets in small molecule drug discovers, determination of binding specificity and stoichiometry, validation of EC50/IC50 values during hit-to-lead, measurement of enzyme kinetics.
What is ITC?
A quantitative study of biomolecular interactions. Directly measures heat absorbed or released.
