Technology & Research

nanoDSF - nano Differential Scanning Fluorimetry.

nanoDSF is a label-free technique that monitors intrinsic protein fluorescence to measure conformational stability.

Summary

When to use nanoDSF.

nanoDSF measures conformational stability, or how well a protein maintains its folded structure under stress. The method tracks how proteins respond to thermal or chemical stress by detecting shifts in tryptophan and tyrosine fluorescence emission.

This provides quantitative stability parameters without requiring external labels or dyes. nanoDSF is critical to gain a complete stability profile for your protein.

nanoDSF is ideal when you need to:

Compare conformational stability across protein variants, buffer conditions, or formulations.

Rank biologics candidates based on thermal or chemical stability.

Detect subtle stability changes that other methods might miss.

Work label-free to avoid artifacts from fluorescent tags.

Screen conditions rapidly with minimal sample consumption.

The science behind nanoDSF.

Protein folding creates distinct fluorescence environments

Proteins fold into three-dimensional structures that bury hydrophobic residues, including tryptophan and tyrosine, in their core. These aromatic amino acids absorb UV light at 280 nm and emit fluorescence, but the wavelength of that emission depends on their chemical environment.

When tryptophan is buried in a folded protein’s hydrophobic core, shielded from water, it emits fluorescence with a maximum around 330 nm. When the protein unfolds and tryptophan becomes exposed to the aqueous environment, the emission maximum shifts to around 350 nm. Tyrosine contributes similarly, though its shift is less pronounced.

This environment-dependent fluorescence shift is the foundation of nanoDSF. By monitoring the ratio of emission at 350 nm to 330 nm (F₃₅₀/F₃₃₀), you can track the folding state of your protein in real time.

Thermal and chemical stress trigger unfolding

During a nanoDSF experiment, proteins are subjected to a controlled stress gradient—either increasing temperature or increasing concentration of a chemical denaturant like urea or guanidinium chloride. As stress increases, proteins begin to unfold. The population shifts from predominantly folded (low F₃₅₀/F₃₃₀ ratio) to predominantly unfolded (high F₃₅₀/F₃₃₀ ratio). Plotting this ratio against temperature or denaturant concentration produces a sigmoidal transition curve. The inflection point of this curve—where the slope is steepest—represents the midpoint of the unfolding transition. This is where 50% of the protein population is folded and 50% is unfolded.

First derivative analysis sharpens the view

Taking the first derivative of the F₃₅₀/F₃₃₀ ratio with respect to temperature (or denaturant concentration) converts the sigmoidal curve into a peak. The maximum of this peak corresponds precisely to the inflection point, making it easier to identify the transition midpoint and compare stability across samples.

Learn more about nanoDSF

Our biophysical tools
that use nanoDSF.

Protein Stability

Prometheus Panta

One experiment. Multiple insights.

Parameters you measure with nanoDSF.

Melting temperature (T_m)

The temperature at which 50% of the protein is folded and 50% is unfolded. Higher T_m indicates greater thermal stability. T_m is the most commonly reported stability parameter and is useful for ranking candidates or conditions.

Onset of unfolding (T_onset)

The temperature where the protein first begins to unfold, identified as the point where the fluorescence ratio starts to deviate from baseline. Some mutations or buffer changes shift T_onset without significantly affecting T_m, making this parameter valuable for detecting subtle stability differences.

Transition slope (sharpness)

The steepness of the unfolding transition, measured as the slope at the inflection point. A steep slope indicates a highly cooperative unfolding process: the protein stays tightly folded until a critical temperature, then unfolds rapidly. A shallow slope suggests a less cooperative, more gradual unfolding.

Chemical denaturation midpoint (C_m or C₅₀)

The concentration of denaturant at which 50% of the protein is unfolded. Like T_m, higher C_m values indicate greater stability.

Free energy of unfolding (ΔG)

The thermodynamic stability of the protein at a given temperature, representing the energy difference between folded and unfolded states. More positive ΔG means the protein is more stable. ΔG compares stability changes between conditions (e.g., wild-type vs. mutant).

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