SPR at the CMI
Surface Plasmon Resonance (SPR) is an optical technique used to measure molecular interactions in real time. SPR can occur when plane-polarized light hits a metal film under total internal reflection conditions. SPR signal is directly dependent on the refractive index of the medium on the sensor chip. The binding of biomolecules results in changes in the refractive index on the sensor surface. In an SPR experiment, one molecule (the Ligand) is immobilized on a sensor chip and binding to a second molecule (the Analyte) is measured under flow. Response is measured in resonance units (RU) and is proportional to the mass on the surface, and for any given interactant, the response is proportional to the number of molecules bound to the surface. Response is recorded and displayed as a sensogram in real time. SPR experiments can be used to measure kinetic binding constants (ka, kd) and equilibrium binding constants (affinity, Ka = 1/Kd).
The CMI has a Biacore T200 from Cytiva, formerly GE Life Sciences.
Applications and Features
- Kinetic binding: ka, kd
- Equilibrium binding: KD
- Macromolecular and small molecule binding
- High sensitivity for small molecule detection
- Robust coupling chemistries
- Temperature Control (4-45C)
- Single Cycle Kinetics
Theory and Design
The Biacore T200 has four flow cells, connected in series. Each experiment uses two flow cells (either flow cells 1 and 2 or flow cells 3 and 4). The 1st is the reference flow cell (with no immobilized ligand). The 2nd is the sample flow cell (with immobilized ligand). SPR signal increases due to accumulation of analyte on the surface and is monitored in real-time.
There are three major steps in an SPR experiment, each requiring optimization:
- Immobilization: the ligand is attached to the sensor chip surface.
- Interaction analysis: the analyte is injected over the sensor surface and binds to immobilized ligand (the association phase) and then analyte is washed off the surface (the dissociation phase).
- Regeneration: the surface is regenerated by removing remaining bound analyte or by removing both ligand and analyte.
Supplies Provided by User:
- Cytiva Biacore Series S sensor chips
- running buffer (should include some detergent, such as 0.05% Tween 20)
all reagents and samples for immobilization and binding assays
- all concentrations of analyte sample should be in running buffer (with fixed concentation of all components, including DMSO, detergent, buffer and salt concentrations)
- pipettors for sample and reagent liquid handling
- (optional) 96-well and 384-well reagent plates and foil
Supplies provided by the CMI:
- plastic tubes and caps (3 sizes)
- instrument cleaning reagents: Desorb 1, Desorb 2, Biadisinfectant
- Purified water for instrument cleaning and running in Standby mode
Biacore T200 Getting Started Handbook, an intro to SPR and tutorial, from Cytiva, formerly GE Healthcare.
Biacore Sensor Surface Handbook, a guide to SPR immobilization strategies, from Cytiva.
Biacore T200 Product Information from Cytiva.
Purchase Sensor chips and reagents from Cytiva.
SPR Pages, a good resource for all things SPR-related.
Additional Biacore T200 instrument and software manuals are available to users on the Biacore T200 computer.