nano Differential Scanning Fluorimetry | nanoDSF
NanoDSF stands for the nano-format of Differential Scanning Fluorimetry (DSF). 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. 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. Additionally, nanoDSF allows for analyzing the colloidal stability of protein solutions (aggregation).
In general, the 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 time-resolution and can reveal even multiple unfolding transitions. 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.
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 monitors the concurrent changes in tryptophan fluorenscence at 330 and 350 nm wavelength.
In order to detect protein aggregation, the special Prometheus NT.48 nanoDSF device available at 2bind features also back-reflection optics. 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. If the protein sample contains aggregated particles, the incident light is scattered by these particles. The loss of reflection intensity is a precise measure for protein aggregation.
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 conformational stability of a protein is described by its unfolding transition midpoint Tm (°C), which is the point where half of the protein is unfolded. 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.
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 monitoring changes in the intrinsic tryptophan fluorescence and aggregation onset temperatures are detected via back-reflection light scattering. The samples can be heated to any temperature in the range 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 with concentrations above 1 mg/ml are required.
Due to the robust detection method and quick analysis pace, nanoDSF is very versatile and can be used in a number of contexts:
- Screening of buffers, formulationd, and buffer additives
- Screening of detergents
- Analysis of thermal and chemical protein unfolding
- 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)
NanoDSF offers a great number of advantages over traditional fluorimetry approaches. Most importantly, in constrast to standard Differential Scanning Fluorimetry, nanoDSF does not require the used of fluorescent dyes like Sypro Orange.
- 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 µg/ml to 200 mg/ml
- Wide molecule size range → From 1 kDa to 1 MDa
FAQ – General
What are typical applications for the nanoDSF Technology? Is screening possible?
NanoDSF is very robust and versatile, because it is a true in-solution, label-free method for analyzing protein stability and protein unfolding. The broad analyzable protein concentration range allows for investigating biopharmaceuticals at very high concentrations, as they are for example used in formulation development.
Furthermore the nanoDSF technology is especially suited for applications in antibody engineering since the ultra-high resolution allows to detect and to analyze multiple unfolding transitions and unfolding events. NanoDSF also provides the possibility to measure the stability of membrane proteins in detergents since this method is truly label-free and does not require any fluorescent dye.
Moreover, due to the fast analysis pace and high throughput, nanoDSF is well suited for screening of buffers, buffer additives or small molecule influence on protein stability and protein unfolding.
How many tryptophans are needed for detection?
One tryptophan per protein is typically sufficient for a measurement due to the high sensitivity of the nanoDSF detector. Also, tyrosin fluorescence can be detected and analyzed. In the latter case, at least 5-10 tyrosines should be present in your protein of interest.
How many samples can be measured in parallel?
With our nanoDSF setup, it is possible to measure 48 samples in parallel
Is it possible to detect wavelength shifts due to protein unfolding?
Since the Prometheus NT.48 detects two different wavelengths, 330 and 350 nm, blue- and redshift of the tryptophan fluorescence can be distinguished.
Are fluorescent dyes required for nanoDSF analysis?
No. NanoDSF is a truly label-free method by monitoring tryptophan and tyrosin fluorescence at 330 nm and 350 nm.
Another label-free method based on tryptophan fluorescence is label-free MicroScale Thermophoresis. This technique allows for measuring steady-state binding affinities of molecular interactions.
How many data points can be recorded?
Due to ultra-high resolution of the nanoDSF NT.48 device, 36000 data points are recorded for an experiment with 48 samples and a heating rate of 1°C/min between 20°C and 95°C.
Is it possible to analyze the stability and unfolding of individual domains in a protein?
The ultra-high resolution of nanoDSF allows us to detect and to analyze multiple transitions and unfolding events.
FAQ – Samples
How much sample is required?
NanoDSF works with ultra-thin glass capillaries. Thus, only 10 µl of sampleis required per experiment.
What concentration range of my samples can be measured?
In general, it is possible to analyze protein solutions from 5 µg/ml up to more than 200 mg/ml.
Is it possible to analyze membrane proteins with nanoDSF?
Yes. Stability of and unfolding of membrane proteins can be analyzed with nanoDSF unlike with other fluorimetry methods, because nanoDSF is truly label-free, works with detergents, and does not require any fluorescent dye which could interfere with the detergent.
Are there any size limitations of my proteins?
No. It is possible to analyze the stability and unfolding of protein complexes up to the Mega-Dalton (MDa) range.
FAQ – Assay Conditions
What is the temperature range for the measurement?
The temperature range spans from 15°C to 95°C. Possible heating rates are from 0.1 to 5°C/minute.
Are there any limitations in buffers or additives?
No. Due to the combination of a label-free setup and the monitoring of tryptophan fluorescence, almost any buffer and any additives, including detergents, can be analyzed in the nanoDSF setup. Even measurements with autofluorescent components and biological liquids are possible to a certain extent, as long as their fluorescence does not intefere too much with tryptophan fluorescence.