Application Notes

Protein Labeling – Improved quantitation of biomolecular interactions by MicroScale Thermophoresis using the RED-NHS 2nd Generation labeling kit

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2 APPLICATION NOTE ©2018 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. be used instead. We currently offer a variety of fluorophores that can be used to label proteins for MST experiments. Interestingly, most commercially available fluorophores for protein labeling are engineered in such a way that makes them suboptimal for TRIC detection since for most fluorescence-based applications a fluorescence signal that is stable and insensitive to environmental changes is prefered. In contrast, the new Monolith Protein Labeling Kit RED-NHS 2nd Generation (Amine reactive), which was specifically developed to be TRIC sensitive, yields higher binding signal amplitudes and improves signal-to-noise (S/N) ratios in MST experiments compared to currently used fluorophores. As a result, more interactions are successfully measured, while using the same convenient labeling chemistry. In addition, the RED- NHS 2nd generation labeling kit provides higher success rates at lower MST power settings without the need of additional time-consuming assay development steps. In this application note, we demonstrate the improved results obtained using the Monolith Protein Labeling Kit RED-NHS 2nd Generation reagents compared to our conventional RED-NHS protein labeling kit. Assay results with enhanced binding amplitudes and up to six-fold higher S/N ratios will be discussed. In addition, data can be analyzed from measurements with lower MST power and shorter MST-on times (data not shown). Introduction MicroScale Thermophoresis (MST) is a biophysical technique that measures the strength of the interaction between two molecules by detecting a variation in the signal of a fluorescently labeled or intrinsically fluorescent target as a result of an IR- laser induced temperature change. The range of the variation in the fluorescence signal correlates with the binding of a ligand to the fluorescent target 1 . Two major factors contribute to the variation in the fluorescence signal: Temperature Related Intensity Change or TRIC, an effect where the fluorescence intensity of a fluorophore is temperature dependent. The extent of the temperature dependence is strongly correlated to the chemical environment of the fluorophore, which can be changed by the binding of a ligand to the target. The other contributing factor to an MST signal is thermophoresis, the effect in which the movement of fluorescent molecules along temperature gradients results in a quantifiable change in their local concentration and the detected fluorescence. The extent of the concentration change depends on the molecule's overall properties including size, charge and conformation or hydration shell. Both TRIC and thermophoresis are influenced by binding events and therefore contribute to the overall measured MST signal 2 . An MST assay requires one of the binding partners to be fluorescently labeled. Alternatively, intrinsic fluorescence from tryptophan and tyrosine amino acids as well as fluorescent fusion proteins can

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