Exclude experimental artefacts
Instead of applying different models in search of the best fit through the data, first, try to optimize the experimental conditions. There are several main groups of experimental artefacts to consider:
Including the instrument, the measuring cell, the matrix on which the ligand is bound, and the buffers.
In short, the system should be clean and stable. Consult the Clean machine item about instrument maintenance. In addition, the instrument must have a stable temperature and be washed and equilibrated properly.
Spikes in the curves can have several origins. Usually, they are caused by small air bubbles in the system. On the other hand, dust particles and aggregates in the solution, IFC or on the detector can cause spikes.
Is the ligand pure and homogeneous? Does the immobilization destroy epitopes? How does it perform after regeneration?
The choice of the ligand can have a great impact on the results. Use pure ligand for direct immobilization and the mildest immobilization technique to preserve ligand integrity. Also, during the experiments, use the mildest regeneration solution and check the ligand activity regularly with a positive control.
Is the analyte pure and interacting only with the ligand? Is it a monomer?
When possible, use a pure analyte to diminish non-specific binding to the sensor surface. Use the right analyte concentration and association and dissociation times to record the full kinetic information of the interaction. Always be aware of rebinding effects or non-ideal kinetics.
Non-specific binding is not always apparent. Therefore, a suitable control in the form of a reference surface is necessary to detect and possibly compensate. To make a matched reference surface is not always straight forward but is essential for proper measurements.