MicroScale Thermophoresis – Technology
2bind uses the innovative MicroScale Thermophoresis Technology (MST) to determine biophysical parameters of molecular interactions. The technique is based on the effect that molecules move in temperature gradients, a physical effect called thermophoresis. The thermophoresis of molecules depends on the molecular size, charge, and hydration shell (Duhr et al. 2006). Since, at least, one of these parameters is typically affected upon binding, the changes in the movement of the molecule in the temperature gradient can be used for the analysis of any kind of bi-molecular interaction or modification of any molecules.
Figure: technical setup MicroScale Thermophoresis and data analysis
(A) An infrared-laser is used to generate a precise focal temperature gradient within a glass capillary, which is filled with the reaction sample. The temperature of the aqueous solution in the laser spot is raised by ∆T = 2 – 5 K. The temperature gradient induces the thermophoretic movement of the molecules in the capillaries. Specific target molecules in the reaction, being located in the infrared laser focus, are monitored by their fluorescence (intrinsic or labeled), and their motion along this temperature gradient is recorded. (B) Initially, fluorescence in the sample is detected in the absence of a temperature gradient to ensure homogeneity of the sample. After 5 seconds, the IR-laser is activated leading to the establishment of the temperature gradient. This causes an initial steep drop of the fluorescence signal – the so-called Temperature- or T-Jump – which reflects the temperature dependence of the fluorophore quantum yield. After the T-Jump, a slower thermophoresis-driven depletion of fluorophores occurs. Once the IR-laser is deactivated, a reverse T-Jump and subsequent backdiffusion of fluorescent molecules can be observed. (C) Since thermophoresis is highly sensitive towards changes in molecular properties, a binding event induces changes in thermophoresis, which can be used to determine equilibrium constants, such as the dissociation constant Kd. For this, a serial dilution of the ligand is prepared, mixed with a constant concentration of labeled target molecule, loaded into capillaries and analyzed in the instrument by subsequent scanning of each capillary. The changes in thermophoresis are then plotted and used to derive the binding constant. The results of a typical binding experiment are illustrated in (C)
The MicroScale Thermophoresis allows to monitor either fluorescent molecules or intrinsically fluorescent proteins (later would be a truly labelfree measurement). The MicroScale Thermophoresis measurements take place in buffers of any kind, even making measurements in serum and lysates possible. The data generation is fast and precise; the data output is comparable to other biophysical methods.
The MicroScale Thermophoresis is the optimal technology to identify and characterize molecular interactions.
For more information about the MST Technology, download the 2bind MicroScale Thermophoresis information booklet.
Or watch this video about MicroScale Thermophoresis.
2bind offers MicroScale Thermophoresis services
We analyze your molecular interactions. Read more on our MicroScale Thermophoresis services