01Identify the two partners and the surface response

Cytiva’s Biacore handbook explains that SPR detects refractive-index changes close to the sensor surface. Accumulation of material near that surface can change the response, allowing an interaction to be followed without fluorescent labelling of the binding partners.Cytiva — Biacore sensor surface handbook (opens in a new tab)

In many SPR reports, ligand means the surface-bound partner and analyte means the partner in the flowing solution. Those experimental roles may differ from the biological use of ligand to mean a molecule binding a receptor.

Write the orientation explicitly. A surface carrying a peptide with a protein in solution is not the same configuration as a surface carrying the protein with a peptide in solution.

Check whether attachment involved a tag, capture reagent or chemical modification. The evidence concerns that presented material, so its relationship to the unmodified molecule should remain visible.

02Read the sensorgram before its fitted constants

For an original hypothetical trace, both the active surface and the reference surface rise when the sample arrives. The difference between them is smaller than the active-surface response alone. That difference matters before the larger trace is described as specific binding.

A reference is a defined comparison, not an automatic guarantee that every background contribution has disappeared. Read what the reference surface contains and whether its response is shown.

Likewise, a sharp change at a solution transition should be interpreted in its experimental context. Its appearance at the same moment as sample introduction does not by itself identify a molecular association rate.

03Check whether the kinetic model describes the observations

Cytiva’s kinetics guide recommends examining fitted curves and residuals, which show differences between observations and model predictions. It also distinguishes interaction kinetics from mass transport: movement of analyte to the surface can influence the observed rate.Cytiva — Kinetics and affinity measurements with Biacore systems (opens in a new tab)

Imagine that a simple model fits the middle of a fictional association trace but repeatedly misses its beginning and the later dissociation region. Those structured deviations deserve explanation even if a software summary reports a small overall error.

Adding a more complex model may improve the fit, but the added mechanism needs a scientific rationale. A better mathematical description does not independently prove the proposed molecular arrangement.

04Compare estimates only with their context attached

Keep the surface orientation, material definition, solution context and analysis model alongside the reported constants. Differences between two publications may reflect these conditions as well as the interaction itself.

Do not treat a larger response as automatically stronger affinity. More material at the surface, different binding capacity or a different analyte mass can affect response magnitude without establishing a tighter interaction.

If the report provides only a final affinity value, note that the supporting curves and model checks were unavailable in the inspected record. Avoid reconstructing them from the number of decimal places.

SPR evidence can support a defined physical interaction under the reported conditions. Establishing downstream receptor signalling or a biological benefit requires evidence addressing those separate outcomes.