APPLICATION NOTE
Protein Labeling
Improved quantitation of biomolecular interactions by MicroScale Thermophoresis
using the RED-NHS 2nd Generation labeling kit.
Tanja Bartoschik, Ivana Bekic, Timm Hassemer, Mariam Mohamadi,
Amit J. Gupta and Nuska Tschammer
NanoTemper Technologies GmbH, Floessergasse 4, 81369 Munich
Abstract
MicroScale Thermophoresis (MST) is a biophysical technique that measures the
strength of the interaction between two molecules by detecting variations in
fluorescence signal as a result of an IR-laser induced temperature change.
TRIC (Temperature Related Intensity Change) together with thermophoresis
contribute to the variation of fluorescence measured. 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.
With the goal of improving the quality and robustness of MST measurements we
selected fluorophores that are more sensitive to temperature changes and are
therefore better reporters of the binding interaction. We show that the use of a
TRIC-optimized Monolith Protein Labeling Kit RED-NHS 2nd Generation boosts the
sensitivity of MST measurements, resulting in improved performance compared
to a previously released kit. In binding affinity experiments the new reagents yield
vastly improved signal-to-noise ratios and binding amplitudes, ensuing higher
quality binding data and in some cases where other fluorophores tested failed,
making binding affinity measurement possible . The kit is offered for our Monolith
systems with red filtersets.
