4 Ways To Reduce Non-specific Binding in Surface Plasmon Resonance Experiments Application note

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During an SPR experiment, there are many experimental parameters that must be controlled in order to optimize the performance of the system and to generate high-quality binding data. Of these parameters, minimizing non-specific binding (NSB) is one of the most imperative as it can directly affect the accuracy of your kinetic data.

SPR experiments typically consist of a ligand – the biomolecule immobilized on the sensor surface, and the analyte – the solubilized biomolecule that binds the ligand. The analyte is flowed over the immobilized ligand, and as they interact there is a change in refractive index at the sensor surface. This change in refractive index is measured as response units (RU), which is then used to calculate the affinity and kinetics of the interaction. In some cases there can be non-specific interactions between the analyte and non-target molecules on the sensor surface, which can inflate the measured RU and lead to erroneous calculated kinetics. This is known as non-specific binding.

Strategies to reduce non-specific binding

One simple preliminary test that can be done is running the analyte over the bare sensor surface without any immobilized ligand. If you observe a significant level of NSB, there are a variety of steps that you can take to limit these interactions. Common strategies include:

Adjusting the pH of your buffer
Using protein blocking additives
Adding non-ionic surfactants
Increasing salt concentration

In order to select the best possible method, it is important to know the characteristics of your analyte and ligand. Knowing the isoelectric point, charge, size, and composition (hydrophilic, hydrophobic) of the molecules can help you determine the most effective condition(s) for reducing NSB in your experiment.