Microscale Thermophoresis (MST)

High-throughput determination of protein affinities using unmodified peptide libraries in nanomolar scale

“…To overcome the inherent caveats of array-based techniques, including immobilization-related artifacts resulting from peptide inactivation or unspecific accumulation of proteins depending on orientation, surface chemistry, and ligand density, we next explored the possibility to measure the same peptide library in-solution using TRIC. Compared with conventional MST setups, the here used TRIC setup allows for reduced sample volumes and is compatible with microtiter plates and thus facilitates higher throughput and automatization ( Gupta et al, 2018 ). Large libraries of unmodified ligands are often analyzed with fluorescently labeled proteins.…”

“…Yet, the FP phenomenon is dependent on ligand and size change upon binding as well as fluorophore and linker characteristics, thus requiring careful assay design ( Zhang et al., 2015 ). In stark contrast, the MST phenomenon is largely independent of molecular weight changes, providing high dynamic range across different assay setups and probe designs ( Gupta et al, 2018 ).…”

“…The nature of the method requires ligand molecules to be non-fluorescent in the investigated red spectral range. Therefore, target molecules are commonly chemically modified with organic fluorophores ( Gupta et al, 2018 ). Here, we explore the use of the Dianthus NT23.PicoDuo (Nanotemper Technologies GmbH) for TRIC measurements in reduced volumes of microtiter plates for affinity determination in unprecedented throughput while maintaining the advantages of conventional MST measurements.…”

Fast Mek1 Hit Identification with TRIC Technology Correlates Well with Other Biophysical Methods

“…For TRIC, an infrared laser is used to induce a highly controlled temperature change by excitation of water molecules. 4,6,7 The correlation between the brightness of the fluorescent probe and the temperature of the solvent is strongly linked to the solvation of the labeled molecule. Therefore, any modification of the physicochemical environment of the fluorophore, for example, a binding event, will modify the observed fluorescence temperature correlation.…”

Development of a Microscale Thermophoresis-Based Method for Screening and Characterizing Inhibitors of the Methyl-Lysine Reader Protein MRG15

“…This temperature dependency is also related to changes in the local microenvironment of the fluorophore, which can be strongly affected by the binding of a ligand to the fluorescent target molecule. 27 From different points of view, MST technology is superior to other methods in determining the parameters of molecular interactions; indeed, it requires low sample consumption, it allows a rapid analysis, no surface immobilization is required, and it allows the measurement of interactions in close-to-native protein conditions. In addition, it enables a live detection of sample aggregation, sticking, and precipitation effects.…”