Analysis Interactions of Macromolecule-Ligand by Isothermal Titration Calorimetry (ITC) (CAT#: STEM-MB-2513-LGZ)

Introduction

Isothermal titration calorimetry (ITC) is a useful tool to gain a complete picture of the thermodynamics of binding reactions. In biological sciences, the interactions between macromolecules are crucial to understanding the mechanisms of cells. Experimental conditions, such as buffers and temperature, can be adjusted according to the specific binding system under study. However, careful planning is required, as certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of macromolecules and ligands need to be measured precisely for reliable results.

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Principle Applications Procedure Materials Notes Related Products

Principle

A reactant is placed in a temperature-controlled sample cell and coupled to the reference cell through a thermocouple loop. The sample cell and the reference cell are in the same external environment. A specific titration agent (selected as required by the test) is added to the sample cell quantitatively. The change in energy in response can be sensitively detected and triggered by a positive or negative feedback thermostat to keep the temperature constant.

Applications

For characterizing thermodynamic parameters of biomolecular interactions.

Procedure

1. Preparation before the experiment.
2. Add 40ul of ligand or small molecule protein solution to the titration syringe, 200ul of large molecule protein solution to the sample pool, and 200ul of distilled water to the reference pool.
3. Set Experimental Parameters in the interface.
4. In the set up interface, set the storage path of experimental data and experimental methods, as well as the current user.
5. Put the titration syringe into the sample pool and click Start to start the experiment. Display real-time images of the experiment on the Real Time Plot interface.

Materials

• Sample Type: protein 50 micrograms, small molecules 10mM/500ul, the higher the sample concentration is the better, protein peptides need to be sent at low temperature.

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