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15 Temperature-Related Intensity Change Temperature-Related Intensity Change (TRIC) technology is a biophysical, in-solution method. It exploits the fact that the fluorescence intensity of a fluorophore is temperature-dependent. This temperature dependency is closely tied to alterations in the fluorophore's local microenvironment, which significantly shi s when a ligand binds to the fluorescent target molecule, changing its shape or flexibility. TRIC should not be confused with MicroScale Thermophoresis (MST), which also uses a temperature increase for visualizing binding events. Despite its co-existence with MST, TRIC is its own powerful technology for detecting binding events. Conclusion TRIC is a 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. How it works TRIC is a fluorescence-based, immobilization- free technology performed in-solution in thin, glass capillaries that hold low, microliter volumes of sample. To analyze an interaction, you label your target molecule with a fluorescent dye and add it to a titration series of the unlabeled ligand. For the measurement, a minute and precise laser-induced temperature increase is applied to the sample. This allows to acquire two fluorescence intensity readings—one at the baseline temperature and another one at a slightly elevated temperature. The K d is calculated by plotting the change in fluorescence against the ligand concentration. Strengths TRIC is an in-solution method in which the binding partners being studied are not immobilized on a biosensor or solid surface. Binding affinity is determined using microliter amounts of sample, and 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. Thanks to the ratiometric analysis of the data, the technique is robust against fluorescence variations from pipetting errors. Beyond K d determination, TRIC is also sensitive to the presence of aggregation, providing additional insights regarding sample quality. A label-free option is available. Because there are minimal moving parts and nothing needs to be cleaned, TRIC can be considered maintenance-free. Weaknesses Absolute binding kinetic constants (k off and k on ) can not be identified with TRIC. While there are label-free options, fluorescent labeling of one of the binding partners is typically required to perform an analysis.

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