heliX®
heliOS Software
The purpose of reference-correction, or referencing for short, is to extract the actual binding kinetics signals from the raw fluorescence signals measured by the heliX® device. Referencing aims to remove any artifacts / side effects such as signal drifts, signal jumps, contributions by unspecific binding etc.
The heliX® device is equipped with two spots allowing for simultaneous measurements. One of the two spots may be used for the actual binding kinetics experiment (“measurement spot”) while the other one serves as a ligand-free reference (“reference spot”). In this configuration, the reference spot signal may be used to reference-correct the measurement spot signal in temporal allocation. This is called real-time referencing.
Blank measurements are control measurements without analyte, conducted before, in between or after actual measurements. Real-time referenced blank signals should appear as flat baseline signals. However, small artifacts may remain. In this case, or in the lack of real-time references, blank signals may be subtracted from the measurement signals for a (second-order) reference-correction. This is called blank referencing.
The combination of real-time referencing and blank referencing is referred to as double referencing.
Ideally, one should start with a scientific hypothesis for the model (e.g. “I expect a basic one-to-one binding process.”). This hypothesis is then tested against the data.
If you have no (good) idea about the process, start with the simplest / most confined model and gradually increase model complexity (number of free parameters) until the model shows acceptable agreement with the data. From a mathematical viewpoint, “acceptable agreement” is defined by goodness of fit statistics, e.g. Χ2-based p-value for nonlinear least squares fitting. Keep in mind that the more complex the model the harder to interpret its parameters.
For kinetics signals, for instance, we recommend starting with a complete-dissociation model that forces the signals back to baseline (at infinity). Only allow for remaining offsets (incomplete dissociation) when the strict model fails.
If you double-click on the fit in your analysis, all used mathematical formulas will be displayed.