Isothermal Titration Calorimetry

Question 0

Attractive features of ITC

Answer 0

• readout is the heat of reaction in solution, so don’t need to label

Question 1

Definition of ITC

Answer 1

Using the heat of complex formation to report on binding interactions

Question 2

Limitations of ITC

Answer 2

• uses lots of material

- has intrinsic sensitivity

Question 3

Instrument used in ITC

Answer 3

VP-ITC micro calorimeter

Question 4

Basic ITC experiment

Answer 4

• 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

Question 5

Initial considerations in ITC

Answer 5

• 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

Question 6

Displacement ITC

Answer 6

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