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Analysis Interactions of RNA and Proteins by Isothermal Titration Calorimetry (ITC) (CAT#: STEM-MB-2499-LGZ)
Introduction
Isothermal titration calorimetry (ITC) is a fast and reliable method for studying the physical basis of molecular interactions. A well-designed experiment can provide a complete thermodynamic characterization of the binding reaction, including Ka, ΔG, ΔH, ΔS and reaction stoichiometry (n). Repeating the experiment at different temperatures allowed the determination of the heat capacity change (ΔCP) of the interaction. Modern calorimeters are sensitive enough to detect even weak biological interactions, making ITC a very popular method among biochemists. While ITC has been used in protein research for many years, it is also being widely used in RNA biochemistry.
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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