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Analysis Interactions of ligand–DNA by Isothermal Titration Calorimetry (ITC) (CAT#: STEM-MB-2507-LGZ)
Introduction
Isothermal titration calorimetry (ITC) is a well-established technique for studying biological interactions. The strength of ITC is that it directly measures the enthalpy change associated with the interaction. Experiments can also generate binding isotherms, allowing the quantification of equilibrium binding constants so that a nearly complete thermodynamic profile can be established. The principles and applications of ITC have been well documented in recent years, and in experiments, the technique is simple to use and, in ideal scenarios, data analysis is trivial.
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.
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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