PR.Panta Control can determine several onsets:

• Onset of unfolding from fluorescence
• Onset of size increase from cumulant radius
• Onsets of aggregation (from scattering intensity and turbidity)

To identify an onset, the software searches for the initial two-state transition in the data. It then fits a two-state transition model to the data, consisting of a linear baseline and a transition region. The onset corresponds to the temperature at which the two-state model fit significantly deviates from the linear baseline. Since the ratio, turbidity, scattering, and cumulant radius data have different magnitudes, the levels at which the deviation becomes significant vary:

For ratio data, this value is set at 0.5%, while for turbidity, it is 0.3%. For cumulant radius and scattering data, the logarithm of the data is used for the fit, with deviations of 5.75% and 1%, respectively.

Figure 1: Unfolding profile of a protein, showing the fluorescence ratio as a function of temperature. Data points are shown in blue. The baseline linear fit (purple line) and the two-state model fit (green line) applied to the data set are shown. The 0.5% deviation between those two fits is automatically calculated, and the corresponding temperature is plotted as the onset (yellow line, ON or Ton).

Onset determination is performed automatically, right after the data is collected in PR.Panta Control. The determined onsets are then stored in the data file. When loading the data into PR.Panta Analysis, this information is retrieved and displayed. In case you are using a version of PR.Panta Analysis that can provide newer, improved algorithms for onset detection, you will be notified and given the option to update.