Overview

The Monolith NT.115 (Nanotemper Technologies Inc.) uses “MicroScale Thermophoresis” (MST) to precisely quantify biomolecular interactions and binding affinity easily and quickly.

MST is a biophysical technique that measures the strength of the interaction between two molecules by detecting variations in fluorescence signal 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.

In addition to MST, temperature increase by the IR laser induces an increased quenching of fluorophores at elevated temperatures. This effect is referred to as a Temperature Related Intensity Change (TRIC). Usually, the fluorescence intensity of a fluorophore is temperature dependent (i.e. intensity decreases with increased temperature) and the extent of the temperature dependence is strongly related to the chemical environment of the fluorophore, which can be affected by the binding of a ligand to the target.

How it works:

A. How the Monolith works: IR laser and fluorescence detection across various samples, contained in glass capillaries.
B. Typical MST curve. There is a drop of fluorescence with IR-laser (temp. increase). Fluorescence returns after IR laser is turned OFF.
C. Binding Curves. The MST signal of a fluorescent molecule changes upon binding to a non-fluorescent ligand.
D. The change in the normalized fluorescence (?Fnorm), which is defined as F1/F0.

Source: Monolith Manual.