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8 MicroScale Thermophoresis MicroScale thermophoresis (MST) quantitatively examines molecular interactions in solution at the microliter scale. It measures binding interactions utilizing the characteristics of thermophoresis combined with Temperature Related Intensity Change (TRIC). Thermophoresis is the movement of molecules in the presence of a thermal gradient and TRIC is the quenching of a fluorophore when subjected to a thermal gradient. Thus, MST is influenced by a molecule's size, charge, hydration shell and the effects of TRIC. Altogether, the data generated results in a precise and robust measurement of binding interactions and modifications. The method works equally well in standard buffers and biological liquids like blood or cell-lysate. MST provides information regarding the binding affinity (K d ) of two or more interacting proteins. How it works MST is performed in-solution in thin, glass capillaries that hold low, microliter volumes of sample. The method is immobilization- free, and analysis can be done in any buffer including complex bioliquids, thus examination occurs in close-to-native conditions. The strength of the interactions between a fluorescently labeled sample (or intrisically fluorescent sample) and a binding partner (or ligand) are measured while a microscopic temperature gradient, induced by an infrared laser, is applied over time. The resulting MST signal is detected and plotted against the ligand concentration to obtain a dose-response curve, from which the binding affinity (K d ) is automatically calculated. Strengths It is an in-solution method in which binding partners being studied are not immobilized on a biosensor or solid surface. With this technology, binding affinity is determined using very small amounts of sample. Results are measured in minutes. It's also very flexible, meaning you can look at molecules of all weights and in all sorts of buffers — ideal for investigating sensitive molecules that need specific buffer conditions, or for looking at interactions in close-to-native conditions. A label-free option is available. Because there are minimal moving parts and nothing needs to be cleaned, MST can be considered a maintenance-free instrument. Weaknesses Absolute binding kinetic constants (K off and K on ) can not be identified with MST. While there are label-free options, fluorescent labeling of one of the binding partners is typically required to perform an analysis. Conclusion MST is the most versatile, rapid and easy- to-use method and can precisely measure binding affinity for virtually every type of biomolecular interaction. It has been shown to obtain results on difficult targets that are challenging to evaluate by other binding affinity methods.

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