Isothermal titration calorimetry: Principles and ...

[Pages:92]Isothermal titration calorimetry: Principles and experimental design

Agenda

Overview of Isothermal Titration Calorimetry ITC experimental design Data analysis Troubleshooting

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What is isothermal titration calorimetry (ITC)

A direct measurement of the heat generated or absorbed when molecules interact

Microcalorimetry offers enhanced information content

Label-free In-solution No molecular weight limitations Optical clarity unimportant Minimal or no assay development

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How Do ITCs Work?

The DP is a measured power differential between

S

R

the reference and sample cells necessary to maintain their temperate difference at close to zero

DP

DT~0

DT

Reference Calibration Heater Sample Calibration Heater Cell Main Heater

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Performing an ITC experiment

Ligand in syringe

Macromolecule in sample cell

Heat of interaction is measured

Parameters measured from a single ITC experiment:

Affinity - KD

Energy (Enthalpy) - DH Number of binding sites - n

Reference Cell

Syringe Sample Cell

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ITC ? Before titration

Ligand ? in syringe Macromolecule in ITC cell

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Titration begins: First injection

Ligand in syringe Macromolecule in cell Macromolecule-ligand complex

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As the first

injection is made,

all injected ligand

is bound to target

macromolecule.

0

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