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nanoDSF Assay Services
nano-Differential Scanning Fluorimetry (nanoDSF) is a biophysical characterization technique used to assess the conformational stability of a biological sample, typically a protein.
Samples are subjected to either temperature ramps or gradients of chemical denaturant, and the intrinsic fluorescence is measured and fit to determine the melting points (Tm).
NanoDSF is a type of Differential Scanning Fluorimetry used to determine conformational protein stability by employing intrinsic tryptophan or tyrosine fluorescence, as opposed to the use of extrinsic fluorogenic dyes that can perturb the system and affect protein stability.
We exclusively use the SUPR-DSF spectrometer from Protein Stable for our DSF measurements.
Reliably measure protein Tm values
The SUPR-DSF instrument measures protein conformational stability via thermal denaturation (10 oC - 105 oC) or chemical denaturation (chaotropes).
Label-Free
nanoDSF utilizes intrinsic protein fluorescence, meaning there’s no need for adding dyes.
Native Sample Measurement
Samples can be analyzed as-is without dilution, rebuffering, or the addition of exogenous fluorophores.
Minuscule Sample Requirements
10 µL of sample is all that’s needed. Sample concentrations as low as 0.1 mg/mL.
Full Spectrum Data
Fluorescence data is recorded from 310nm-420nm for every sample, enabling global data fitting.
High-Throughput
Measure 384 samples or replicates per experiment. We pass the value and cost savings onto you.
What sets Ichor’s nanoDSF services apart?
Ichor’s nanoDSF services include sample handling, standard assay execution, data analysis & reporting, and development of custom workflows for unique projects.
Ichor exclusively uses the SUPR-DSF from Protein Stable which analyzes samples in low-volume 384-well fluorescence plates, as opposed to other instruments that utilize low-throughput and expensive capillaries. We pass the savings and efficiency on to you.
Ichor provides full workflow support, including protein production, sample preparation and processing, nanoDSF assay execution, and statistical analysis complete with a full report deliverable.
The SUPR-DSF records full-spectrum fluorescence data from 310 nm to 420 nm, enabling calculation of the Barycentric Mean (BCM) of the fluorescence waveform as the protein unfolds. BCM, coupled with derivative analysis, produces better results than the intensity and ratio methods used by competing instruments by taking advantage of the entire spectrum.
We offer a range of orthogonal biophysical techniques, including Circular Dichroism (for more detailed structural and thermodynamic analysis) and Surface Plasmon Resonance (for binding studies).
Small Molecule Binding Studies
Ichor’s nanoDSF services go beyond routine formulation screening experiments and into the realm of small molecule binding studies. Traditional DSF assays are very limited in their tolerance of small molecules due to the constraint of only measuring two emission intensities at 330 nm and 350 nm. If a small molecule has an autofluorescent contribution to these two filter sets, an accurate shift in the Tm will not be measurable. The SUPR-DSF uses a CMOS fluorimeter which gives us emission intensity between 310 nm and 420 nm, making it possible to choose a range that is free of interference.
Data Analysis with the SUPR-DSF
Barycentric Mean Fluorescence Analysis
Barycentric Mean: BCM measures the center of mass of the fluorescence waveform, enabling the computation of wavelength shifts as the protein unfolds. BCM produces better results than the ratio method by taking advantage of the entire fluorescence spectrum.
Melt Curve Analysis
The data that results from the BCM fluorescence analysis can be fit using either a thermodynamic model or by taking the 1st Derivative and calculating the inflection points.
Featured Capabilities / Case Studies – Courtesy of Protein Stable
Formulation Ranking – Case Study 1
Finding Formulations Faster with the SUPR-DSF
- Screened mAb Infliximab and 96 formulations in triplicate in a single experiment using the FORMOscreen®
- 24 Formulations improved stability of the first transition by more than 1.0°C.
- Top stabilizer, shown as the red circle, improved the stability of both the first (2.3°C) and second transition (1.3°C).
- Methodology is readily adaptable to forced-degradation studies.
Formulation Ranking – Case Study 2
Targeting Optimal Buffers for Downstream Crystallization Screening
- The process of X-ray crystallography requires purified protein to form crystals in optimal buffer conditions on specialized crystallization plates.
- A primary buffer screen of larger global parameters (buffer system, pH, salt, concentration etc.) before this experimental step can greatly reduce the window of buffers to test and minimize consumable costs.
- The stability of proteins in initial buffer conditions has been directly linked with crystal formation success in subsequent crystallography screens.
- Thermal stability of Beta-Lactoglobulin was screened against a series of buffer formulations in RUBIC Buffer Screen.
- Optimal conditions were found to be formulations with high salt, low pH citric acid.
- nanoDSF can also provide other critical parameters such as Tonset & ΔH.
- Conclusion: A quick formulation buffer screen can help to narrow in on promising conditions for single crystal growth, saving weeks of time.
Protein Characterization & Engineering
Speeding Up Early Stage Biotherapeutic Discovery with Next Generation nanoDSF
- Screened 15 mutants of parent protein (WT).
- Identified 8 mutants that improved stability by > 2°C better than WT – Mutants 01, 07 & 08.
Identification of AAV Serotypes and Characterization of Their Stability
AAVs for Gene Therapy: Simultaneously Determine Component Serotypes and Optimize Stability
- AAVs have different amino acid sequences and capsid structures and therefore different Tm In this study, AAV2, AAV5, & AAV6 were compared.
- AAVs exhibit sharp melting points, allowing easy differentiation.
- nanoDSF makes formulation optimization easy.
- A low concentration of 2×1012 VP/mL is sufficient to accurately measure AAV stability.
- KBI Biopharma (Louisville, CO) sourced all the AAVs, performed the sample preparation, and acquired data with the SUPR DSF.
Frequently Asked Questions
Learn more about Ichor’s nanoDSF services by reading below.
Traditionally, techniques such as Differential Scanning Calorimetry (DSC) and Circular Dichroism (CD) were used to assess protein thermal stability, however they suffer from the drawbacks of high sample consumption and low throughput. In contrast, nanoDSF uses exceptionally small quantities of sample (typically 1-10 µg/well) and up to 384 conditions can be fully screened in a single 1.5 hour experiment.
Our end-to-end nanoDSF services include novel nanoDSF assay development. Assays can be developed around your specific protein of interest using either a thermal or chemical denaturant gradient. Commercially available formulation libraries can be employed, and similarly small molecule ligand binding can be investigated.
Because nanoDSF is a technique based on the intrinsic fluorescence of proteins, the only prerequisite for samples is that they contain one or more tryptophan or tyrosine residues. Typical sample concentrations span a broad range from approximately 0.1 mg/mL to 250 mg/mL.
The most common nanoDSF applications are 1) Formulation Ranking, 2) Ligand Binding, and 3) Protein Engineering. Other common applications include identification and stability characterization of AAV serotypes.
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.
You will receive all raw and processed data produced by the instrument. This includes the plate map and sample preparation info, raw fluorescence data as a function of temperature and wavelength, fitted melt curves, and a PDF report containing all melting temperatures and related thermodynamic data.
The actual cost will depend on the scope and complexity of the project. Assays are typically conducted and billed per 384-well plate, meaning that you the client receive significant value for submitting large numbers of samples for analysis.
