SPR & BLI

Analyte flows through microfluidic channels over a gold sensor chip (e.g. CM5 dextran). Very sensitive and well suited to detailed kinetics, but needs careful referencing and regeneration.

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Surface plasmon resonance (Biacore)

Analyte flows through microfluidic channels over a gold sensor chip (e.g. CM5 dextran). Very sensitive and well suited to detailed kinetics, but needs careful referencing and regeneration.

How it differs from the standard method

Amine coupling at pH below the ligand's pI.
Keep Rmax low (~50–150 RU) for kinetics.
Flow 30–100 µL/min to avoid mass transport.

In this lesson · about 1 min read

Watching binding happen in real time
From curves to constants
Artefacts that look like binding
Key terms
When to use it
Sub-techniques

1Watching binding happen in real time

Both methods immobilise one partner (the ligand) on a sensor surface and flow or dip the other (the analyte) over it. As analyte binds, mass builds up at the surface and an optical signal changes.

Surface plasmon resonance

Light reflected from a gold film under the sensor chip: the resonance angle shifts as the refractive index next to the gold changes. 1 RU ≈ 1 pg/mm² of protein.

nm shift

Bio-layer interferometry

White light reflected from two layers at the tip of a fibre-optic biosensor: binding thickens the layer and shifts the interference pattern.

2From curves to constants

For a simple 1:1 interaction, dR/dt = ka·C·(Rmax − R) − kd·R. Each concentration gives a curve whose association rate (kobs = ka·C + kd) and dissociation rate (kd) are fitted globally across the series.

3Artefacts that look like binding

Bulk refractive-index jumps from buffer mismatch: square steps at the start and end of each injection. Removed by subtracting a reference surface.
Mass-transport limitation: with too much ligand or too little flow, analyte binds faster than it is delivered, so curves look linear and sluggish.
Incomplete regeneration: analyte left from previous cycles raises baselines and lowers capacity.
Too short a dissociation: if the signal barely drops, kd (and so KD) is undefined.

Key terms

Practise these as flashcards →

Ligand / analyte

Ligand is immobilised on the surface; analyte is in solution.

ka (kon)

Association rate constant, M⁻¹s⁻¹.

kd (koff)

Dissociation rate constant, s⁻¹; complex half-life = ln2 / kd.

Equilibrium dissociation constant = kd/ka, in M. Lower means tighter binding.

Rmax

Maximum response if every ligand site is occupied; scales with ligand density and analyte/ligand mass ratio.

Double referencing

Subtracting both a reference surface and buffer-only injections to remove bulk effects and drift.

When to use it

Ranking antibodies, nanobodies or drug candidates by affinity and off-rate.
Measuring kinetics label-free with small sample amounts.
Checking that a purified protein is active (binds its partner).

Compared with alternatives

Isothermal titration calorimetry →

Gives KD, stoichiometry and enthalpy in solution without immobilisation, but no kinetics and much more sample.

Microscale thermophoresis →

KD in solution with tiny volumes; no on/off rates.

ELISA →

Endpoint, labelled, high-throughput; doesn't give kinetics.

Fluorescence polarization →

Solution binding of a small labelled ligand; good for competition screens.

Sub-techniques

Choosing one here switches the whole page, including the protocol and the lab, to it.

Analyte flows through microfluidic channels over a gold sensor chip (e.g. CM5 dextran). Very sensitive and well suited to detailed kinetics, but needs careful referencing and regeneration.

Amine coupling at pH below the ligand's pI.
Keep Rmax low (~50–150 RU) for kinetics.
Flow 30–100 µL/min to avoid mass transport.