14
1.
Introduction to MicroScale Thermophoresis
(MST)
MicroScale Thermophoresis
TM
(MST) provides an easy, fast and precise approach to quantify
biomolecular interactions. It allows for the detection of a wide range of interaction types, ranging
from ion and fragment binding up to interactions of macromolecular complexes such as liposomes
or ribosomes [1-3]. The main key feature of MST is the capability to work under close-to-native
conditions: Measurements are performed immobilization-free and in any buffer, or even in complex
bioliquids such as serum and cell lysate. In addition, the method combines straight forward sample
preparation, low sample consumption of less than 4
μl per sample at nM concentration, and a large
dynamic range with dissociation constants from the sub-nM to mM range. The NanoTemper
Technologies Monolith
®
instruments are maintenance-free, have low running costs and use an
intuitive software user interface with provides guided assay development and automated data
analysis, making MST a one-of-a-kind, low-hurdle biophysical technology for every lab.
1.1. Method
The Monolith instruments analyze MST signals for rapid and easy determination of affinity constants.
MST is based on the generation of a locally restricted and highly precise temperature change in a
sample by infrared (IR) laser light, which cannot be achieved by conventional heating procedures
using e.g. thermal elements. Changes in sample fluorescence upon activation of the IR laser are
monitored to characterize and quantify binding events. The response of the sample fluorescence
upon IR laser activation is based on distinct physical principles which are described in the following
section.
1.1.1. Underlying physical principles
MicroScale Thermophoresis (MST) is a biophysical technique that measures the strength of the
interaction between two molecules by detecting a variation in the fluorescence signal of a
fluorescently labeled or intrinsically fluorescent target as a result of an
IR-laser induced
temperature change
. The range of the variation in the fluorescence signal correlates with the
binding of a ligand to the fluorescent target.
Two major factors contribute to the variation in the fluorescence signal:
1- TRIC (
Temperature Related Intensity Change
) - An effect where the fluorescence intensity of a
fluorophore is temperature dependent. The extent of the temperature dependence is strongly related
to the chemical environment of the fluorophore, which can be changed by the binding of a ligand to
the target.
2-
Thermophoresis
-An effect where the movement of fluorescent molecules along temperature
gradients results in a quantifiable change in their local concentration and therefore of the observed
fluorescence. The extent of the concentration change depends on the molecule’s overall properties
like size, charge and conformation.
Both TRIC and thermophoresis are influenced by binding events and therefore contribute to the
overall recorded MST signal.
