Prometheus NT.Plex characterizes thermal and chemical unfolding under native conditions using nanoDSF technology. Absolutely label-free, nanoDSF precisely measures the intrinsic fluorescence of a protein while it’s being subjected to either chemical or thermal denaturation.
nanoDSF uses high quality capillaries for a number of reasons — only microliters of sample are required, a wide range of concentrations are quickly measured in-solution, multiple conditions are assessed in one run, and hard-to-measure viscous samples are examined with ease. Just dip a capillary into a sample, place it in Prometheus, and hit start to get Tm, Tonset and Tagg results in minutes. And, if Cm, ΔG and ΔΔG are needed, those can be done too with the same system.
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)
Samples per run: Up to 24 manually
Sample volume measured: 10 μL
Detected molecule concentration range: 0.005 – 250 mg/mL (standard IgG)
Experiment time per run – thermal unfolding (from 1 °С/min up to 7 °С/min ramp): 18 – 75 minutes
Experiment time per run – chemical unfolding: 1 minute
Precision of 1 °С/min thermal ramp: ± 0.2 °С
- Ramping options: 0.1 °С/min to 7 °С/min
- Temperature range: 15 °С – 95 °С (at 25 °С ambient temperature)
Fluorescence detection: 330 nm and 350 nm
Dimensions: 35 cm W x 51 cm H x 52 cm D
Weight: 30 kg
- Aggregation detection optics
- High temperature upgrade 15 °С – 110 °С (at 25 °С ambient temperature)
Take assumptions out of the equation — Get reproducible, quantitative results early on from discovery to validation and production. Use Prometheus to generate precise unfolding temperatures (Tm and Tonset), critical denaturant concentrations (Cm), free folding energy (ΔG and ΔΔG), and aggregation onset (Tagg) from start to finish. Be confident about moving forward with the right choice every step of the way.
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
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 26 chips 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.
Thomas Clairfeuille, Alexander Cloake, Daniel T. Infield, José P. Llongueras, Christopher P. Arthur, Zhong Rong Li, Yuwen Jian, Marie-France Martin-Eauclaire, Pierre E. Bougis, Claudio Ciferri, Christopher A. Ahern, Frank Bosmans, David H. Hackos, Alexis Rohou, Jian Payandeh. Science, vol.363, 6433 (2019)
Prakash Rucktooa, Robert K. Y. Cheng, Elena Segala, Tian Geng, James C. Errey, Giles A. Brown, Robert M. Cooke, Fiona H. Marshall & Andrew S. Doré. Scientific Reports, vol.8, 41 (2018)
Severin Wedde, Dr. Christian Kleusch, Dr. Daniel Bakonyi & Prof. Dr. Harald Gröger. ChemBioChem, vol.18(24), p.2399–2403 (2018)
Cyrille Garnier, François Devred, Deborah Byrne, Rémy Puppo, Andrei Yu. Roman, Soazig Malesinski, Andrey V. Golovin, Régine Lebrun, Natalia N. Ninkina & Philipp O. Tsvetkov. Scientific Reports, vol.7, 6812 (2017)
Goncalves L, Kracher D, Milker S, Rudroff F, Fink M, Ludwig R, Bommarius A, Mihovilovic M. Advanced Synthesis & Catalysis, 359, p.2121–2131 (2017)
Shukla E, Agrawal S, Gaikwad S. International Journal of Biological Macromolecules, vol.98, (Mtcc 5185) p.387-397 (2017)
David Gervais, Justin Hayzen, Charlotte Orphanou, Alexandra McEntee, Christine Hallam & Rossalyn Brehm. Enzyme and Microbial Technology, vol.98, p.26-33 (2017)
Shalev Gihaz, Diána Weiser, Adi Dror, Péter Sátorhelyi, Moran Jerabek-Willemsen, Dr. László Poppe, Dr. Ayelet Fishman. ChemSusChem, vol.9, p.3161–3170 (2017)
Rune Busk Damgaard, Jennifer A. Walker, Paola Marco-Casanova, Neil V. Morgan, Hannah L. Titheradge, Paul R. Elliott, Duncan McHale, Eamonn R. Maher, Andrew N.J. McKenzie & David Komander. Cell, vol.166, p.1215-1230 (2016)
Mehmood S, Corradi V, Choudhury H, Hussain R, Becker P, Axford D, Zirah S, Rebuffat S, Tieleman D, Robinson C, Beis K. Journal of Biological Chemistry, vol.291 (41), p.21656-21668 (2016)
nanoDSF: [2bind.com] (date of treatment: 13.08.2019)