nano Differential Scanning Fluorimetry | nanoDSF
Intrinsic tryptophan fluorescence of proteins is strongly dependent on their 3D-structure and hence the local surroundings of the tryptophan residues. Using chemical denaturants or a thermal gradient, proteins can be unfolded, which leads to changes in their intrinsic tryptophan fluorescence. This translates into fluorescence emission peak shifts and intensity changes. NanoDSF monitors these fluorescence changes with high resolution and can reveal even multiple unfolding transitions. NanoDSF is therefore highly applicable in antibody engineering, membrane protein characterization and protein quality control.
The figure illustrates the principle behind thermal protein unfolding: Increasing temperature causes tryptophan residues becoming solvent exposed upon unfolding of the three-dimensional protein structure. NanoDSF monitors the concurrent changes in tryptophan fluorenscence at 330 and 350 nm wavelength.
In order to detect protein aggregation, the Prometheus NT.48 nanoDSF device features also back-reflection optics. Normally, visible light passes through the capillaries containing the protein sample of interest without any interference and is reflected by a mirror on the capillary tray and then quantified by the detector. If the protein sample contains aggregated particles, the incident light is scattered by these particles. The loss of reflected intensity is a precise measure for protein aggregation.
The conformational stability of a protein is described by its unfolding transition midpoint Tm (°C), which is the point at which half of the protein is unfolded. The truly labelfree 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.
Up to 48 capillaries are filled with 10 µl of protein sample and simultaneously scanned at 330/350 nm wavelengths. Melting temperatures are recorded by the intrinsic tryptophan fluorescence and aggregation onset temperatures are detected via back-reflection light scattering. Within 75 min, the samples are heated from 25°C to 95°C. Importantly, samples can be studied without the use of a dye and with free choice of buffer and detergent. Melting temperatures of proteins with a concentration between 5 µg/ml and 250 mg/ml can be analyzed. In order to obtain high quality aggregation onset temperatures, protein solutions commonly with concentrations above 1 mg/ml are required.
NanoDSF is a rapid and accurate method for studying the conformational and colloidal stablity of proteins and is thus perfectly suited for protein engineering, formulation development, and screening approaches.
- Screenings of buffers, formulation, and buffer additives
- Screening of detergent influence
- Long-term protein and antibody storage optimization
- Forced-degradation stability testing
- Comparison of biosimilar proteins and antibodies with respect to stability and aggregation
- Batch-to-batch comparison assays
- Deep feature analysis (influence of mutations, modifications, conjugations on protein stability and aggregation)
- Low sample consumption (Only 10 µL of sample required)
- Free choice of assay buffers (Also biological liquids possible such as serum or cell lysate and other additives/detergents)
- Very short analysis time (enables high throughput)
- Optimal data quality and resolution (Dual 350/330 nm UV-detection)
- Wide temperature range (Analysis possible from 15°C to 95°C)
- No labeling required (Close-to-native analysis possible)
- Wide concentration range (5 µ/ml to 200 mg/ml)
- Wide molecule size range (From 1 kDa to 1 MDa)