Microscale Thermophoresis involves the following steps:
One of the binding partners is labeled using standard fluorescent labeling protocols. NanoTemper provides blue, green or red dyes optimized for protein compatibility and MST analysis.
A titration series of up to 15 dilutions is prepared, where the concentration of the fluorescent binding partner is kept constant and the concentration of the unlabeled (i.e. non fluorescent) molecule is varied.
After incubation time sufficient for the reaction to reach equilibrium (e.g. 5 min.), the reaction is transferred into a glass capillary. The capillary is placed on the sample tray, which can accommodate up to 16 capillaries, and the sample tray is placed in the instrument.
Filled capillaries are scanned consecutively and the thermophoresis signal of each sample is measured. The analysis software is used to calculate the dissociation coefficient.
Microscale Thermophoresis can monitor the binding of single ions (40Da) or small molecules (300Da) to a target as well as the binding of ribosomes (2.5MDa). The capillary format is easy to handle and offers maximum flexibility in the experiment scale. The capillaries come with different surface coatings to stabilize even the most complex samples in solution. Capillary volume is 5 μl. Labeling kits contain fluorescent dyes that are widely tested with MST. The labeling protocol ensures good labeling efficiency and purification. The fluorescence emission and detection fits perfect to the BLUE and RED channel in the NT.115 instruments.
The Monolith NT.115 and NT.LabelFree are NanoTemper´s instruments for MST measurements. The Monolith NT.115 measures thermophoresis of fluorescently labeled molecules. 3 models of the Monolith NT.115 instrument are offered, which differ in the excitation/detection spectrum to detect blue, green and/or red fluorescent dyes, as defined by the respective color of the excitation light. The Monolith NT.LabelFree measures thermophoresis by intrinsic tryptophan fluorescence. The Monolith NT.LabelFree instrument has an excitation wavelength of about 280nm and an emission wavelength around 360nm. Both instruments are equipped with the standard sample tray and can process automatically up to 16 samples per run in 10 minutes.
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