Signal-to-noise ratio
The signal-to-noise ratio is used to evaluate the quality of Fnorm data. It is defined as the response amplitude divided by the noise of the measurement.
For Binding Check and Buffer Screen experiments, this simply means dividing the Response Amplitude by the standard deviation of replicates.
For Binding Affinity experiments, this means calculating the following:
Where ri denotes the residual of the fit at a given data point and ŕ the average of all residuals. The number of data points is given by n. A signal-to-noise ratio of more than 5 is desirable, while more than 12 reflects an excellent assay.
Recommendations to increase the signal-to-noise ratio
- Check whether all prerequisites for the investigated system are met. For example, dif- ferent types of biomolecules (Antibodies, GPCRs, Membrane Proteins, Nucleic Acids, Kinases, Enzymes) have different requirements for their functionality, such as Reducing Agents, co-factors, buffer restrictions and additives.
- Change the Protein Labeling strategy, for example by altering the labeling buffer and thus the location of the fluorophore on the protein. Also, switching the labeling kit (e.g. from RED-NHS 2nd Generation to RED-NHS) can improve assay quality.
- Change the Assay Buffer. A change in buffer composition, ion strength or pH can influence MST behavior significantly.
- Reverse the setup (switch target and ligand).