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12 Conclusion SPR is a highly sensitive and precise tool that provides affinity and kinetic data. Due to its long tenure, SPR is widely used for both academic and industry research applications, and has many established standard protocols. However, it can be costly and a bit complicated to operate, and is not suited as a walk-up instrument in a dynamic research setting. Surface Plasmon Resonance Surface plasmon resonance (SPR) is a common biosensor-based technique for measuring biomolecular interactions. How it works SPR measures the amount of mass bound to a biosensor surface. It requires the immobilization of one binding partner, the ligand, to this biosensor (o en called chip). The other binding partner, the analyte, is injected into a flow cell and comes into contact with the ligand at a constant flow rate. This increase in mass bound to the surface results in a signal change. The resulting sensograms display an association phase where the analyte is captured by the ligand as a function of the association rate constant of the interaction (k on ). This is followed by a dissociation phase, where only buffer is pumped through the flow cell and the analyte molecules detach as a function of the dissociation rate constant (k off ). By testing different analyte concentrations presented to a constant amount of immobilized ligand, the k on , k off and K d of the interaction can be determined. Strengths SPR determines binding kinetics in addition to binding constants. It allows for higher-sensitivity measurements and conserves lab resources by consuming low volumes of sample. Since SPR has been around for a while, standard protocols exist for a number of molecules and interactions. If you work on one of these well-characterized molecules or interactions, SPR can be a great tool. Weaknesses Immobilization of the ligand is not always straightforward. Although diverse chip surfaces, coupling chemistries and even specific chips are available, some ligands cannot be immobilized in a functional way. Many "difficult" targets, such as membrane proteins, are fragile and do not tolerate the acidic surface of the SPR assay. Another weakness are the microfluidic tubes used to create the sample flow. They need to be flushed regularly and thoroughly to remove unspecific binding and avoid clogging. Also, in order to save time and money, researchers re-use chips for several binding experiments. For interactions where the analyte does not wash off easily, scientists must identify regeneration conditions that can break the ligand-analyte interaction but leave the ligand intact, which can be difficult. Additionally, since SPR measures changes in refractive index, any buffer component that influences the refractive index of a sample (e.g. DMSO, a common solvent for small molecules) can cause artifacts in SPR measurements. Usually, control experiments are necessary when working with DMSO. Finally, because SPR depends on mass changes on the chip surface, this technology is not an optimal solution for small molecule research due to the small change in mass.