Surface Plasmon Resonance is a phenomena generated by the incidence of polarized light onto an electrically conductive surface at the interface between it and a solution with different bulk refractive indices. This will only occur if the incident light contacts the surface at such an angle as to cause total internal reflection. Part of the light will be reflected while another part of the light will be absorbed by the electrons on the surface and propagate as an electric field across the surface. This electric field is known as the evanescent wave. The angle of the light such that a minimum amount of reflected light is observed is known as the resonance angle and is directly proportional to refractive index of the solution adjacent to the surface. Biacore instruments measures the resonance angle and the variations in this angle caused by changes in refractive index of the solution. As mass binds to the surface, it modifies the bulk refractive index of the solution causing the angle of the reflected light minima to change leading to a measurable signal. The amount by which the resonance angle changes is directly proportional to the mass bound. Molecular interactions are thus studied by immobilizing one binding partner onto the gold sensor surface (known as the ligand) and using these surfaces to capture the other binding partner from solution (known as the analyte).
There are a wide variety of experiments possible with the Biacore, some of which include:
- Kinetics Analysis of Molecular Interactions
- Concentration Measurement
- Activity Assays
- Binding Site Analysis
- Screening of Interaction Partners
Instrument Info: Biacore3000 (Biacore, Installed Sep. 2000)
Temperature Range: 20-30°C (Instrument is generally run at 25°C)
Each user is required to use their own Sensor Chip for their experiments and these may either be purchased from Biacore directly or from the facility. These sensor chips come with a variety of surface chemistries suitable for specific applications and allow for the immobilization of ligand through a wide variety of coupling chemistries. A careful literature search is generally the best method to determine what concentration of sample will be required for both the ligand and the analyte as this will vary greatly from experiment to experiment. Generally concentrations in the 0.1-1mg/mL are needed for the ligand and uM or nM concentrations for the analyte depending on the strength of the interaction.
Training involves the completion of one experiment under the supervision of Core Facility personnel. The experiment can be one of the trainee’s choosing.To get a basic feel for the procedure of the experiment, it is recommended that you review the tutorial before coming in to use the instrument.