⚖️ Buffer Matching

SPR and GCI instruments sense refractive index (RI) in the evanescent volume near the sensor surface. Any difference in RI between your sample and running buffer produces a step artefact — an abrupt jump at injection start and a matching drop at injection end — that is entirely unrelated to binding. This is the bulk shift.

The magnitude can be large. A 1% DMSO mismatch produces roughly 1,460 RU of bulk shift on a Biacore-class SPR instrument (~10⁶ RU per RIU). A typical small-molecule binding response is 5–50 RU. One percent of mismatch can therefore dwarf the signal you are trying to measure. The calculator below lets you explore this relationship quantitatively. Note: RU values assume ~10⁶ RU/RIU (Biacore-class SPR); GCI instruments report pg/mm² or their own response units on different sensitivity scales, and BLI is largely insensitive to bulk RI.

First-line defence — reference subtraction. Running a reference flow cell (or reference well) and subtracting its signal removes the matched component of the bulk shift. Subtracting a buffer-blank injection from matched analyte injections (double referencing) removes any residual drift. Reference subtraction should always come first; buffer matching and solvent correction address what remains after it. See the Reference Subtraction page for details.

Buffer matching. The cleanest mitigation is eliminating the mismatch entirely: dilute your analyte directly into the running buffer, or exchange both into a common buffer by dialysis or spin-column. For compound libraries, pre-spot DMSO into the running buffer at the concentration used in the analyte samples, then dilute compounds into that matched buffer. Run a solvent-correction calibration series before and after your kinetic experiments.

Solvent correction. When a residual DMSO mismatch cannot be eliminated — common with diverse compound libraries — solvent correction is applied. It is a calibration curve built from 5–8 injections of running buffer spiked with bracketing DMSO concentrations (spanning the range in your analyte samples), run on the reference flow cell only. The instrument uses this curve to model the residual reference-subtracted bulk response and subtract it from each analyte injection. Solvent correction cannot fully compensate for mismatches beyond ~0.5–1% DMSO because the RI–response relationship becomes nonlinear. For a step-by-step protocol, see the DMSO Correction page.

Recognising the artefact. A bulk-shift artefact appears as a square step — identical in magnitude on the active and reference flow cells — that rises instantly at injection start and drops instantly at injection end, returning the signal to baseline with no exponential tail. Large salt mismatches additionally perturb local binding kinetics (ionic-strength effects on k_on), not just RI.