MICROSCALE THERMOPHORESIS

Short Description:

Measure changes of mobility of the molecules in microscopic temperature gradients to determine binding affinities

Long Description:

Microscale Thermophoresis (MST) is a powerful new technology to quantify biomolecular interactions in a few microliter solution. The MST method 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. Virtually any molecule (small molecules, DNA, RNA, proteins, peptides, sugars, lipids, ribosomes etc) can be analyzed. The thermophoresis is detected and quantified using either covalently attached or intrinsic fluorophores. For example, the thermophoresis of a protein typically differs significantly from the thermophoresis of a protein–ligand complex due to binding-induced changes in size, charge and solvation energy. For deriving binding constants, multiple capillaries with constant concentrations of protein and increasing concentration of ligand are scanned consecutively and thermophoresis is detected. The analysis software is used to plot and fit the change in thermophoresis to yield a Kd. This technology has several advantages over other standard techniques to analyze interactions, such as surface plasmon resonance (SPR) and isothermal microcalorimetry (ITC). It can measure affinities in free solution without surface immobilization with low sample consumption and within sub-nM to mM range.  Experiments can be carried out with a broad range of solution conditions, including detergent mixtures and complex bioliquids.

Device  : NanoTemper´s Monolith NT.115 BLUE/RED model

Data output : Titration curves and Kd values

User Guide:

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.