FAQs

What is nanoDSF, and how does it differ from traditional DSF techniques? 

• NanoDSF is a variant of Differential Scanning Fluorimetry that measures protein stability by monitoring intrinsic fluorescence from tryptophan and tyrosine residues during thermal or chemical denaturation. Unlike traditional DSF techniques that rely on exogenous dyes, which can introduce dye-protein interactions and disrupt stability, nanoDSF eliminates this risk. This makes it more accurate, reliable, and compatible with a broad range of proteins, including those in sensitive or complex formulations. 

What is the difference between Thermal Shift/Melt Assays, (nano)DSF, and Thermofluor? 

• A Thermal Shift Assay (a.k.a. Thermal Melt Assay) measures the shift in the melting temperature (Tm) of a biomolecule such as a protein under varying conditions, such as buffer formulation, drug concentration, or mutations. The Tm is the temperature at which 50% of the molecules in solution exist in a denatured state. There are various techniques for conducting thermal shift assays.  
• Thermofluor is a type of thermal shift assay, typically conducted on a qPCR instrument, that indirectly measures the melting point of proteins via the addition and temperature-dependent association of an extrinsic hydrophobic turn-on fluorophore dye to the internal hydrophobic regions of a protein as it unfolds.  
• nanoDSF is a subset of thermal shift assays that measures the melting point of proteins via the intrinsic fluorescence of native tyrosine and tryptophan residues. nanoDSF is a sensitive, label-free technique that allows for direct measurement under native conditions and formulations.  
• A conceptually and technically similar type of assay is the chemical melt assay, which measures the concentration of chemical denaturants (e.g., guanidine-HCl) that induce 50% denaturation. 

What are the core applications of the SUPR-DSF? 

• The SUPR-DSF is used to investigate protein conformational stability via experiments involving either thermal or chemical denaturation. The most common SUPR-DSF applications are 1) Formulation Ranking, 2) Ligand Binding, and 3) Protein Engineering. Other common applications include identification and stability characterization of AAV serotypes. At the centre of each of these applications is protein conformational stability

What are the advantages of using nanoDSF over DSC or Circular Dichroism (CD)? 

• Traditional methods like Differential Scanning Calorimetry (DSC) and Circular Dichroism (CD) assess protein thermal stability but require high sample volumes and offer relatively low throughput. In contrast, nanoDSF instruments like the SUPR-DSF use minimal sample amounts (1-10 µg/well) and can screen up to 384 conditions in just 1.5 hours, providing a high-throughput, efficient solution for protein stability analysis. 

Why choose the SUPR-DSF over other nanoDSF instruments? 

• The SUPR-DSF stands out from other instruments with its unbeatable combination of affordability, efficiency, and advanced performance. By utilising inexpensive, commercially available consumables and offering free software, it drastically lowers operational costs. Its 384-well capacity provides 4x higher throughput, maximizing productivity for busy labs. Additionally, the SUPR-DSF’s full-spectrum fluorescence analysis (310-420 nm) ensures precise measurements and interference-free results, delivering unmatched accuracy for protein stability studies. Choose SUPR-DSF for smarter, faster, and more cost-effective analysis. 

What sample types can be analysed by the SUPR-DSF? 

• The SUPR-DSF relies on the intrinsic fluorescence of proteins, requiring only that samples contain one or more tryptophan or tyrosine residues. It’s suitable for a wide range of proteins, including those in complex formulations or with varying stability profiles. 

What is the concentration range for samples in SUPR-DSF experiments? 

• The SUPR-DSF accommodates a broad concentration range, typically from 0.1 mg/mL to 250 mg/mL. The minimum concentration needed for sufficient signal-to-noise depends on the protein's primary sequence. 

How much total sample is required for a typical experiment? 

• The SUPR-DSF uses exceptionally small sample volumes, requiring only 1-10 µg per well (sequence dependent), making it highly efficient and ideal for limited or precious samples. 

Does the SUPR-DSF require any special dyes or reagents? 

• No, the SUPR-DSF leverages the intrinsic fluorescence of proteins, eliminating the need for exogenous dyes or specialised reagents that could interfere with stability measurements. 

Can the SUPR-DSF be used to study small molecule binding interactions? 

• Yes, the SUPR-DSF is well-suited for small molecule binding studies. Its full-spectrum fluorescence detection and barycentric mean (BCM) analysis of the entire fluorescence waveform avoids or minimizes interference from autofluorescent compounds, ensuring accurate results even in challenging experimental conditions. 

Does the protein need to have specific residues for nanoDSF analysis? 

• Yes, the protein must contain one or more tryptophan or tyrosine residues, as the intrinsic fluorescence from these residues is fundamental to the technique.  

What is the throughput of the SUPR-DSF? 

• The SUPR-DSF offers high-throughput capabilities, analysing up to 384 samples, conditions, or replicates in a single thermal ramp experiment.

How long does a typical SUPR-DSF experiment take? 

• A complete experiment on the SUPR-DSF, such as a thermal ramp from 15–105 oC at a ramp rate of 1 oC/min., takes approximately 1.5 hours. Therefore, with automation, approximately 16 experiments (6,144 samples) can be completed in one day on a single instrument.

What type of consumables does the SUPR-DSF require? 

• The SUPR-DSF uses affordable, commercially available 384-well qPCR plates and plate seals, avoiding expensive proprietary consumables: 
• Appropriate microplates are readily available from commercial suppliers: 

• Bio-Rad Laboratories: Hard-Shell® PCR Plates, thin wall, skirted, black/black; part Number: #HSP3866; suitable for 10 µL - 30 µL working volumes 

• Azenta Life Sciences: FrameStar® 384 Well Skirted PCR Plate; part number: 4ti-0386; black wells, black frame; suitable for 10 µL - 30 µL well volumes 

• Seals are readily available from third party vendors including:  

• Azenta Life Sciences: qPCR Adhesive Seal; part number: 4ti-0560; optically clear adhesive film, pressure activated adhesive; compatible with all 384-well PCR plates 

Why do the qPCR plate seals need to be used? 

• The qPCR plate seals are optically transparent to near-UV light. They contain a silicone-based pressure-sensitive adhesive encapsulated in microspheres that is only activated on the rim of each well during seal application. This prevents both contamination of the sample and dramatically reduces the fluorescence background of the measurements.  

Does the SUPR-DSF software require a separate license or additional cost? 

• No, the SUPR-DSF software (SUPR-Suite) is provided free of charge, ensuring no hidden fees or ongoing licensing costs. You may install the software on an unlimited number of machines at your institution.  

What kind of data can I obtain from a SUPR-DSF experiment? 

• The SUPR-DSF generates full-spectrum fluorescence data as a function of temperature, which allows for the generation of melting curves and precise onset (T_onset) and melting temperatures (T_m). These insights provide detailed information about protein stability and unfolding mechanisms. All raw and processed data and results can be exported in .csv files for additional optional processing or plotting in 3rd party software. Alternatively, the SUPR-Suite software effortlessly generates beautiful .pdf data reports.   

What is the wavelength range for fluorescence detection in the SUPR-DSF? 

• The SUPR-DSF excites samples with a high power (10 mW) 280 nm LED laser and detects fluorescence across a broad range of 310-420 nm with a CMOS assay spectrometer, allowing for full-spectrum analysis and flexibility in avoiding interference. 

Can the SUPR-DSF be used for chemical denaturation studies? 

• Yes, the SUPR-DSF supports chemical denaturation experiments in addition to thermal ramps, enabling versatile protein stability analysis. 
• If your primary focus is chemical denaturation experiments, it is important to consider the strengths of both the SUPR-DSF and SUPR-CM to determine the best fit for your needs. While the SUPR-DSF can perform chemical melts, the SUPR-CM is specifically optimized for this application and offers several advantages for dedicated chemical stability studies: 
• The SUPR-CM supports both standard black 96- or 384-well SBS plates, offering more flexibility in plate selection compared to the SUPR-DSF, which is limited to 384-well PCR plates.  

• Additionally, the SUPR-CM does not require plate seals and allows for longer integration times of up to 1000 ms, enabling lower protein usage and lower background signal. In contrast, the SUPR-DSF must be run with a seal and at lower integration times due to background signal from the seal. 

• However, the SUPR-DSF does include temperature control, allowing it to handle both thermal and chemical melts, making it more versatile if you plan to explore thermal stability in addition to chemical denaturation.  
• If your needs are exclusively focused on chemical melts, the SUPR-CM is the ideal choice for achieving higher-quality results with more flexibility and efficiency. On the other hand, if your research requires the ability to perform both thermal and chemical melts, the SUPR-DSF remains the best choice. 

What temperature range and ramp rates are supported by the instrument? 

• The SUPR-DSF can conduct temperature ramps from 10-105 oC with ramp rates of 0.1-10 oC/min. The instrument is also capable of running isothermal experiments (e.g., incubations or chemical melts).  

What fluorescence integration times are supported by the instrument? 

• The SUPR-DSF supports integration times of 1-25 ms for standard thermal ramp experiments; if in doubt, we recommend starting with 25 ms as this rarely needs to be adjusted. Integration times of up to 100 ms are possible in isothermal experiments.