Protein Labeling – Improved quantitation of biomolecular interactions by MST using the His-Tag Labeling Kit RED-tris-NTA 2nd Generation

With the goal of improving the quality and robustness of MicroScale Thermophoresis (MST) measurements, we enhanced our protein labeling kits by selecting fluorophores that are more sensitive to temperature changes (Temperature Related Intensity Change or TRIC) and are therefore superior reporters of binding events.

MST is a biophysical technique that measures the strength of the interaction between two molecules by detecting variations in fluorescence signal measured from one of the binding partners 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. TRIC is an effect where the fluorescence intensity of a fluorophore is dependent upon temperature changes and binding interactions. Thermophoresis is the quantifiable movement of fluorescent molecules along a temperature gradient. Both TRIC and thermophoresis contribute to the variation of fluorescence measured by MST.

This TRIC-optimized generation of dyes boosts the sensitivity of MST measurements and yields vastly improved binding amplitudes and signal-to-noise ratios. Incorporated into the 2nd Generation kits, the RED-tris-NTA 2nd Generation dye, just like the RED-tris-NTA dye, binds efficiently and selectively to polyhistidine-tags and has minimum impact on the biochemical and physicochemical attributes of the protein or peptide. Additionally, the high affinity and selectivity of the dye for His-tags enables the labeling of target proteins even in crude
cell lysates. Combining the advanced properties of the 2nd Generation dye with the merits of a site specific and purification-free labeling approach further broadens the range of interactions accessible with MST and simplifies the way to better results.