Analysis Interactions of Nanoparticle–Protein by Isothermal Titration Calorimetry (ITC) (CAT#: STEM-MB-2509-LGZ)

Introduction

Isothermal titration calorimetry (ITC) is a complementary technique that can be used to study the adsorption of proteins on nanomaterials, as it quantifies the thermodynamic parameters of in situ interactions between molecules. Once a nanomaterial enters a biological medium, a protein corona forms around the nanomaterial, which can greatly affect the surface properties of the nanomaterial and thus its behavior. ITC enhances our understanding of nanoparticle - protein interactions because it provides information on binding affinity (binding constant Ka), interaction mechanism (binding enthalpy ΔH, binding entropy ΔS, and Gibbs Free energy ΔG), and binding stoichiometry n.

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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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