The unfolding profile of a protein is a plot of its fluorescence signal against temperature. The protein is heated at a defined, constant ramp over a defined temperature range. Both ramp and range depend on user settings. During the heating process, fluorescence is recorded at 330 nm and 350 nm. Fluorescence is plotted against temperature, typically by plotting the fluorescence ratio 350 nm / 330 nm (see image below, right panel). The resulting unfolding profile is then automatically analyzed for inflection points (IP). By fitting the data in PR.Panta Analysis, Tms (melting points) can also be derived.

The left panel shows a protein's fluorescence emission spectra at a given temperature. For most proteins, the native (folded) state shows its emission maximum around 330 nm (purple), while the unfolded state shows its emission maximum around 350 nm (blue). During the unfolding process, the maximum is shifted, and in most cases, the overall fluorescence intensity decreases. These two wavelengths therefore provide all the information needed to follow protein unfolding via fluorescence.

In Prometheus software, unfolding profiles can be visualized in different ways, including:

• Single wavelength. This means the fluorescence values plotted against temperature are those recorded at 330 nm OR 350 nm.
• Fluorescence ratio (F350 nm / F330 nm). This means the fluorescence values plotted against temperature are the ratio values. The ratio has the advantage of canceling out potential effects of fluorescent buffer additives or air bubbles and is therefore more robust and universally useful than the single wavelength data. However, the single wavelength data can sometimes provide additional information. 
• First derivative. The first derivative is an available option for ratio and single wavelength data. The first derivative contains no additional information but can be easier to read since unfolding events are displayed as peaks (positive or negative) instead of transitions, which can be easier to identify visually.
• Data points. This simply shows the data points, as recorded during the experiment. Zoom in using the mouse wheel to see individual points.
• Smoothed curve. This option uses an internally developed smoothing process to show a curve instead of individual data points.
• Points and curve. As the name says, this shows both data points and smoothed curve at the same time.