Microscale Thermophoresis is an easy, fast and precise way to quantify biomolecular interactions. It measures the motion of molecules along microscopic temperature gradients and detects changes in their hydration shell, charge or size. It allows measuring a wide range of biomolecular interactions under close-to-native conditions: label-free, immobilization-free, in any buffer or complex bioliquid. NanoTemper's unique technology is ideal for basic research applications requiring flexibility in the experimental scale, as well as for pharmaceutical research applications including small molecules profiling.
An infrared-laser is used to generate precise microscopic temperature gradients within thin glass capillaries that are filled with a sample in a buffer or bioliquid of choice. The fluorescence of molecules is used to monitor the motion of molecules along these temperature gradients. The fluorescence can be either intrinsic (e.g. tryptophan) or of an attached dye or fluorescent protein (e.g. GFP).
To detect the thermophoretic mobility in a MST experiment, one of the interaction partners needs to be fluorescent. In case of the Monolith NT.LabelFree (and the LabelFree detector in the NT.Automated), the intrinsic tryptophan fluorescence of proteins can be exploited and no modification is required. For the other detectors, one of the interactants must be fluorescently labeled. Several coupling chemistries are available but the primary amine labeling with an N-hydroxysuccinimide (NHS) ester is one of the most commonly used for labeling proteins. NanoTemper Technologies offers labeling kits optimized for MST experiments. Alternatively, proteins can be expressed as fusion proteins (e.g. GFP), and biomolecules such as DNA and RNA oligos or peptides can be purchased from different companies carrying site-specific fluorophores.
Since MST measures the interaction at equilibrium, the interactants must be titrated against each other. The dilution series need to cover the baseline with fully unbound molecules and saturation of fully bound complex. This means that the dilution must be at least 20 fold below and above the dissociation constant. In the serial dilution, the labeled compound is kept constant and the binding partner is titrated.
After the making the dilution series the capillaries are filled with 4 µl of each of the samples.
The capillaries are placed in the capillary tray and inserted in the instrument. The first scanning (the so-called “Cap Scan”) will locate the exact position of the capillaries and detect the overall fluorescence in the capillaries. To pinpoint the capillary position is essential to ensure correct focusing of the fluorescence detection and laser excitation with µm precision in the center of each capillary.
After the Cap Scan, the MST experiment can be conducted. In the figure the IR-laser is switched on after 5 seconds baseline measurement. Two effects happen when the laser is switched on: 1) a fast temperature jump (time scale ~ 1s) and the thermophoretic movement (timescale > 10s).
In a typical binding experiment, all 16 capillaries are measured within 10 min. For data analysis, the average thermophoretic value of each curve is plotted against the concentration of the binding partner resulting in a dose-response curve. The points are fitted and the K Dof the interaction is determined.
MST experiments are performed to measure binding affinities, but the MST data and also the fluorescence signal recorded during the Cap Scan can provide information on sample property and homogeneity. These signals can be easily identified, thus assisting you to quickly optimize assay conditions: