SPR detects binding events by measuring changes in refractive index at a gold sensor surface. When molecules bind, the mass increase shifts the angle of reflected light, which we monitor in real time. This label-free technique lets you watch binding happen as it occurs, not just measure an endpoint. The result is a sensorgram: a curve showing association, equilibrium, and dissociation phases.

CM5 (carboxymethyl dextran) is the workhorse for most protein applications using amine coupling. NTA chips are ideal for His-tagged proteins; SA chips for biotinylated molecules; and L1 chips for lipid-based or membrane protein work. Your choice affects immobilization chemistry, surface capacity, and potential non-specific binding. Match the chip to your molecules properties.

One RU corresponds to approximately 1 pg/mm² of mass on the sensor surface. It's a measure of how much material is bound. Higher molecular weight analytes produce larger RU responses at the same molar concentration. Typical kinetic experiments aim for 10-100 RU of analyte binding to balance signal quality against mass transport artifacts.

The equilibrium dissociation constant (K) describes the overall strength of binding, lower K values mean tighter binding. The association rate constant describes how quickly molecules bind, while the dissociation rate constant divided by the association rate constant. Interestingly, two molecules with identical K can have very different kinetic profiles tat meaningfully affect drug behavior.

In direct binding, your ligand is covalently attached to the sensor surface; in capture assays, you first immobilize a capture molecule (like anti-Fc antibody or streptavidin) that grabs your ligand non-covalently. Capture approaches let you use fresh ligand for each cycle and control orientation, but add complexity and potential for ligand dissociation during measurement. Choose based on your ligand's stability and whether orientation matters.

SPR isn't ideal when neither partner can be immobilized without losing function, when you need thermodynamic parameters (use ITC), when kinetics are outside instrument range (extremely fast or extremely slow), or when you need to measure binding in specific cellular contexts. Very weak interactions (>mM) can also be challenging due to signal-to-noise limitations.

Yes. SPR is widely accepted for characterizing biopharmaceuticals, and binding kinetics data regularly appears in IND, BLA, and NDA submissions. The key is defensible methodology, appropriate controls, and clear documentation of your analytical approach. Biacore data in particular has extensive regulatory precedent.