APPLICATION NOTE
Lea Martin, Sascha Schwarz & Dennis Breitsprecher
NanoTemper Technologies GmbH
Flößergasse 4
81369 Munich, Germany
Abstract
A detailed analysis of protein stability is a prerequisite for both, the basic understanding
of protein folding mechanisms as well as for the successful development of biologicals in
the pharmaceutical industry. Here we demonstrate the performance of the Prometheus
NT.48 instrument, which detects intrinsic protein fluorescence changes upon thermal or
chemical unfolding of up to 48 samples in parallel.
Keywords: Protein stability, enzyme buffer screen, formulation and thermal unfolding
Introduction
The assessment of protein thermal stability is an integral part in basic research, drug
discovery and drug development [1]. For instance, shi s in the melting temperature (T
m
)
of a target protein upon binding to a small molecule ligand are routinely used in primary
screens in the drug discovery process [2]. In addition, the thermal and chemical stability
of biologicals, such as antibodies, is o en monitored to establish optimal conditions
for large-scale production and long-term storage [3, 4]. Moreover, the careful analysis
of protein unfolding and refolding mechanisms can yield important insights into the
thermodynamic origins of protein folding, thereby helping to elucidate the molecular
basis of degenerative diseases such as Alzheimer, Parkinson or diabetes [5].
The basis of label-free fluorimetric analysis of protein folding lies in the properties of
the fluorescent amino acid tryptophan. Since tryptophan is a hydrophobic amino acid,
it is mostly located in the hydrophobic core of proteins where it is shielded from the
surrounding aqueous solvent. Upon unfolding however, tryptophan is exposed, which
Prometheus: the platform for analyzing protein stability
and thermal unfolding of proteins
