Measuring Interactions Using Microscale Thermophoresis

Traditionally biomolecular interaction analysis relies on methods such as binding experiments, pulldown experiments or EMSAs for example, or the use of a biophysical method such as ITC or SPR. However, all of these methods have limitations. MicroScale Thermophoresis or MST is a technique that allows you to obtain binding constants for virtually any type of biomolecular interaction in a close-to-native environment. This means it is possible to measure interactions for everything from protein-protein complexes to small molecule binding. MST can overcome some shortcomings of traditional methods because it measures biomolecular interactions using just a few µl of sample and has almost no limitation in molecule size, affinity or buffer. MST measurements can even be performed on complex bioliquids such as cell lysates.

So how does it work? The technology is based on the fact that molecules move within temperature gradients; this is called Thermophoresis. This movement depends on a number of factors including the hydration shell, charge and size of molecules. MST uses fluorescence to follow the movement of the molecules; this fluorescence can either be intrinsic to the molecule (by measuring the fluorescence of tryptophan for instance) or an attached dye or fluorescent protein, such as GFP. This makes MST very sensitive and accurate, and even label-free in cases of intrinsic fluorescence. 

So, how do you set up an MST experiment?  First, you prepare a dilution series of your ligand and add a constant amount of the fluorescent molecule. The sample is filled into capillaries and loaded into the instrument. The instrument uses an IR laser to generate a temperature gradient and the thermophoresis of fluorescent molecules in this gradient is monitored. Then, software plots the thermophoresis signal against the concentration of your ligand, generating a binding curve. In as little as 10 min you can obtain a dissociation constant for your interaction.

A major advantage of MST is that you can measure binding affinities without any immobilization. You can investigate binding between virtually any type of molecule using methods that don’t rely on size changes upon binding. Assay optimization is rapid and straight forward, with low sample consumption compared to other biophysical methods.  MST is a versatile, fast and accurate way to study and quantify biomolecular interactions.

Watch Video:

Related Products from: NanoTemper Technologies