![]() 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. 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. Correcting for this puts Andromeda on a collision course with the Galaxy with a closing speed of around 100 km/s.NanoDSF stands for the nano-format of Differential Scanning Fluorimetry (DSF). This line of sight velocity of the centre of mass of Andromeda is incredibly precise, but this includes the motion of our Sun around our Galactic centre. A more modern value is $-301 \pm 1$ km/s ( van der Marel
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