The depicted spectra are only for visualization.
#BLUESHIFT E REDSHIFT FULL#
Please note that the Prometheus NT.48 nanoDSF device does not measure full fluorescence spectra, but only monitors at the two distinct wavelenghts 330 nm and 350 nm. This leds to a change of their fluorescence properties towards higher wavelenghts. A red-shift is the most common result of protein denaturation, because the tryptophan residues, which are usually packed inside the hydrophobic core of the protein are now exposed to the hydrophilic solvent environment. To make life easier, below is a guid of the most common cases of nanoDSF data analysis and interpretation.įirst, let us consider a case where the denaturated state shows a fluorescence spectrum that is red-shifted (dotted lines) in relation to the spectrum of the native state (solid line). The interpretation of nanoDSF data can range from easy and straightforward to relatively complex, because the change in fluorescence if often a complex mixture of changes in fluorescence intensity at different wavelengthts as well as shifts of the underlying fluorescence spectra of the denatured protein in relation to the native state. In order to obtain high quality aggregation onset temperatures, protein solutions with concentrations above 1 mg/ml are required. Melting temperatures of proteins with a concentration between 5 µg/ml and 250 mg/ml can be analyzed.
#BLUESHIFT E REDSHIFT FREE#
Importantly, samples can be studied without the use of a dye and with free choice of buffer and detergent. The samples can be heated to any temperature in the range from 25☌ to 95☌. Melting temperatures are recorded by monitoring changes in the intrinsic tryptophan fluorescence and aggregation onset temperatures are detected via back-reflection light scattering. Up to 48 capillaries are filled with 10 µl of protein sample and simultaneously scanned at 330/350 nm wavelengths. The truly label-free nanoDSF technique monitors the intrinsic tryptophan fluorescence of proteins, which is highly sensitive for the close surroundings of the tryptophan residues and which changes upon thermal unfolding. The conformational stability of a protein is described by its unfolding transition midpoint T m (☌), which is the point where half of the protein is unfolded.
NanoDSF is a differential scanning fluorimetry method able to analyze the conformational stability and colloidal stability (aggregation behavior) of proteins under different thermal and chemical conditions. The loss of reflection intensity is a precise measure for protein aggregation. If the protein sample contains aggregated particles, the incident light is scattered by these particles. Normally, visible light passes through the capillaries containing the protein sample of interest without any interference, is reflected by a mirror on the capillary tray, and finally quantified by the detector. In order to detect protein aggregation, the special Prometheus NT.48 nanoDSF device available at 2bind features also back-reflection optics. NanoDSF monitors the concurrent changes in tryptophan fluorenscence at 330 and 350 nm wavelength. The figure above illustrates the principle behind thermal protein unfolding: Increasing temperature causes unfolding of the three-dimensional protein structure and thus tryptophan residues to become solvent exposed. NanoDSF is therefore highly successful in antibody engineering, membrane protein characterization, protein quality control, buffer screening, protein unfolding analysis, and small molecule compound binding screening. NanoDSF monitors these fluorescence changes with high time-resolution and can reveal even multiple unfolding transitions. This translates into fluorescence emission peak shifts and intensity changes. Using chemical denaturants or a thermal gradient, proteins can be unfolded, which leads to changes in their intrinsic tryptophan fluorescence. In general, the intrinsic tryptophan fluorescence of proteins is strongly dependent on their 3D-structure and hence the local surroundings of the tryptophan residues. Additionally, nanoDSF allows for analyzing the colloidal stability of protein solutions (aggregation). Consequently, nanoDSF is a great tool for buffer and formulation screening as well as screening of small molecule compound libraries for influence on protein stability and shifts of thermal melting temperature. It is a fast, robust, high-quality, and – most importantly – label-free and in-solution method for the analysis of protein stability, thermal protein unfolding and melting temperature analysis. NanoDSF stands for the nano-format of Differential Scanning Fluorimetry (DSF).
0 kommentar(er)
