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Analysis Interactions of ligand and Target Molecules in Drug Development by Isothermal Titration Calorimetry (ITC) (CAT#: STEM-MB-2510-LGZ)

Introduction

An important goal of drug development is to design inhibitors and ligands with high binding affinity to target molecules. When optimizing these interactions, the precise determination of binding affinity becomes increasingly difficult once nanomolar levels are approached and exceeded. Isothermal titration calorimetry (ITC) can be used to determine the complete binding thermodynamics of a ligand down to the picomolar range by using an experimental mode called displacement titration.




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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