The three different ways to present Spectral Shift/MST data are outlined directly below. They are all available in the MO.Control software (Monolith X) or MO.Affinity Analysis software Compare Results view (Monolith NT.115, NT.115Pico, NT.LabelFree, NT.Automated). More information on how to present and describe MST data in publications is shown below.

Ratio (Spectral shift) or normalized fluorescence (Fnorm in ‰, MST)

Here, the ratio (Spectral shift, Figure 1A) or Fnorm (MST, FIgure 1B) is plotted against the concentration of the ligand. This is how Spectral Shift/MST data is presented without further processing. This presentation is suited to present a single binding curve, but it is not the best way to illustrate multiple binding curves in a single graph. The curves will start at different ratio/F_norm values and/or will show different amplitudes, which can make comparing them difficult.

Figure 1A. Ratio without normalization

Figure 1B. Fnorm without normalization

Baseline corrected ratio (Spectral shift) or Fnorm (MST)

In this normalized display, the change in ratio or Fnorm is plotted against the concentration of the ligand. To obtain ΔRatio (Spectral Shift) or ΔFnorm (MST), the baseline value is subtracted from all data points of the same curve. Thus, by definition, ΔRatio or ΔFnorm is 0 in the unbound state and can adopt positive or negative values, depending on the direction of the response amplitude. This allows the comparison of both the amplitude of the binding curve as well as the K_d of multiple experiments in one graph. When the amplitudes are very different, the response curve with the smaller amplitude might mistakenly be interpreted as a non-binder. In case of very different amplitudes, use Fraction Bound.

Figure 2A. Ratio without normalization

Figure 2B. Fnorm without normalization

Fraction bound

Here, the fraction bound is plotted against the ligand concentration. All ΔRatio or ΔFnorm values of a curve are divided by the curve amplitude, resulting in the fraction bound (from 0 to 1) for each data point. This approach is independent of both the starting level and the amplitude, and thus allows for comparing K_ds of interactions with very different amplitudes. However, any further information about the binding amplitude is lost.

Figure 3. Fraction bound

General remarks on publishing Spectral shift or MST data

• The x-axis should have a logarithmic scale.
• Fluorescence traces should be displayed as inset or in the supplemental material for MST experiments. 
• MST-on times should be displayed in the MST traces or can be mentioned in the figure legend.
• Perform biological replicates, e.g. at least 3 independently pipetted measurements.