Isothermal Titration Calorimetry Flashcards Preview

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Flashcards in Isothermal Titration Calorimetry Deck (7):

Definition of ITC

Using the heat of complex formation to report on binding interactions


Attractive features of ITC

- readout is the heat of reaction in solution, so don't need to label


Limitations of ITC

- uses lots of material
- has intrinsic sensitivity


Instrument used in ITC

VP-ITC micro calorimeter


Basic ITC experiment

- fill upper syringe with ligand at high conc
- fill lower reservoir with protein at lower conc
- titre small aliquots of ligand into protein
- subtract appropriate blank titrations to control for heats of dilution


Initial considerations in ITC

- critical concentrations of macromolecule and ligand
- choice of buffer
- dialysis
- degas all solutions prior to loading
- reference cell
- sample cell
- titrant concentration (molar ration = 2 after final injection)
- heat of dilution of titrant


Displacement ITC

High affinity ligand is titrated into protein that is prebound to a weaker inhibitor