Surface Plasmon Resonance (SPR)

The Biacore T200 is a instrument that measures Surface Plasmon Resonance (SPR) and is used to measure molecular interactions in real time. In an SPR experiment, one molecule (the Ligand) is immobilized to 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 on a sensogram in real time.  SPR experiments can be used to determine the kinetics (association and dissociation rate constants, kon and koff) and affinity (equilibrium binding constants, KA and KD) of molecular interactions.

The CMI has a Biacore T200 from GE.

For information on access fees, policies and getting started at the CMI, see the CMI Access Page.

SPR Experimental Phases

There are three major phases in a Biacore experiment and each will need to be optimized for you experiment.

Immobilization

  • the Ligand is attached irreversibly or reversibly to the sensor chip surface

Interaction analysis

  • In the association phase, the Analyte is injected over the sensor surface
  • In the dissociation phase, the Analyte is washed off the surface

Regeneration

  • the surface is regenerated by removing remaining bound analyte or by removing ligand (which will also remove remaining analyte)

SPR Theory

Surface Plasmon Resonance (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 on a sensogram in real time.  

Supplies Provided by User:

  • GE 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
  • pipettors, tips and tubes for sample and reagent liquid handling
  • (optional) 96-well reagent plates

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

Getting Started Guides and Tips:
GE Biacore T200 Getting Started Handbook, an intro to SPR and tutorial.
CMI Biacore T200 Getting Started Guide, tips for getting started on the Biacore T200.

Other Resources:
GE Biacore Sensor Surface Handbook, a guide to SPR immobilization strategies.
Biacore T200 Product Information from GE Healthcare.
Purchase Sensor chips and reagents from GE Healthcare.  

SPR Pages, an excellent resource for all things SPR-related.

Biacore T200 instrument and software manuals are available to users on the Biacore T200 computer.