Affinity. Stability. Conformation.

Piotr Wardega

NanoTemper Technologies, GmbH, Flößergasse 4, 81369 München, Germany


MicroScale Thermophoresis (MST) is a powerful technique to quantify  biomolecular interactions. It is based on thermophoresis, the directed movement of molecules in a temperature gradient, which strongly depends on a variety of molecular properties such as size, charge, hydration shell or conformation. Thus, this technique is highly sensitive to virtually any change in molecular properties, allowing for a precise quantification of molecular events independent of the size or nature of the investigated specimen. When performing a MST experiment, a temperature gradient is induced by an infrared laser. The directed movement of molecules through the temperature gradient is detected and quantified using either covalently attached or intrinsic fluorophores. By combining the precision of fluorescence detection with the variability and sensitivity of thermophoresis, MST provides a flexible, robust and fast way to dissect molecular interactions.

nanoDSF is our advanced Differential Scanning Fluorimetry technology. It detects smallest changes in the fluorescence of tryptophan present in virtually all proteins. The fluorescence of tryptophans in a protein is strongly dependent on its close surroundings. By following changes in fluorescence, chemical and thermal stability can be assessed in a truly label-free fashion. The dual-UV technology by NanoTemper allows for rapid fluorescence detection, providing an unmatched scanning speed and data point density. This yields an ultra-high resolution unfolding curves which allow for detection of even minute unfolding signals. Furthermore, since no secondary reporter fluorophores are required as in conventional DSF, protein solutions can be analyzed independent of buffer compositions, and over a concentration range of 250 mg/ml down to 5 µg/ml. Therefore, nanoDSF is the method of choice for easy, rapid and accurate analysis of protein folding and stability, with applications in membrane protein research, protein engineering, formulation development and quality control.

The surface acoustic wave technology (SAW) allows for an in-depth analysis of molecular interactions in real time. Binding kinetics can be precisely determined by detecting mass and binding-induced conformational changes. In addition to standard interactions, viscous, colored and turbid samples can be analyzed, and also complex samples including membrane preparations can be investigated. The SAW technology measures changes in mass and conformation separately, thus providing new insights to mechanisms of binding in addition to the binding kinetics (kon, koff and the dissociation constant Kd) and stoichiometry. SAW is based on the precise detection of the properties of surface acoustic waves that travel along the biosensor. Upon interaction with molecules on the sensor surface, distinct characteristics of the acoustic waves are altered; changes in total mass on the biosensor result in a shift of the wave's phase providing information about the on- and off-rates, as well as the stoichiometry of the interaction. Simultaneously a change in flexibility of the molecules alters the wave's amplitude. This directly reflects changes in the conformation of the molecules, e.g. after binding to compounds.Both signal types are detected and quantified separately, and can be used to comprehensively characterize the interaction mechanism of the molecules on a kinetic and structural level.