The NanoTemper Prometheus NT.48 device characterizes nanoDSF, meaning thermal unfolding, chemical denaturation and aggregation of proteins with up to 48 samples in a single run. nanoDSF is a modified version of Differential Scanning Fluorimetry (DSF) to determine to determine protein stability on the basis of intrinsic fluorescence of the aromatic amino acids tryptophan and tyrosine, which are present in most proteins.
The stability of protein is in principle measured by thermal unfolding, where a linear temperature gradient is set to native proteins. The thermal stability of a protein is typically described by the so-called melting temperature Tm, where 50% of the protein molecules is unfolded. Protein unfolding leads to the acceptability of the aromatic amino acids to the outside, so that fluorescence at 350 nm and 330 nm increases. Protein stability e.g. increases when a protein is located in an optimal buffer or when a specific ligand is bound (Fig. 9). It can therefore be used to screen for optimal buffer conditions for any specific protein or antibodies, and for screening for not yet known ligands for proteins in “Prometheus” device. The intensity of backreflected incident light can be used for measuring protein aggregation since large particles reduce backreflection.