Instruments available: BIAcoreT200
The original T100 was part of the BBSRC Systems Biology Centre
Equipment location: New Biochemistry 00-064
Equipment coordinator: Dr David Staunton
Equipment charge: £150/day
Surface Plasmon Resonance
Surface plasmon resonance is a powerful tool for measuring molecular interactions without the requirement for a labelled molecule. One of the molecules (the ligand) is immobilized on the surface of a ‘sensor’, while the other (the analyte) is free in solution and passed over the surface. Association and dissociation can be measured in real time. The technique uses a thin film of gold coated onto a glass slide. When polarised light of a specific wavelength is passed through the glass most of it is reflected at the gold layer, but at a certain well defined angle interactions with the free electrons in the metal absorb energy and cause a shadow in the reflection. The precise angle of this shadow is affected by the refractive index of the medium close (<300 nm) to the gold layer. Interactions which cause a change in refractive index in this evanescent wave can then be detected and measured. The Biacore system employs a gold layer coated with dextran and enclosed in a flow cell so that samples dissolved in a suitable medium can be passed over the surface. The dextran is available in many derivatised forms and can be activated by standard affinity chromatography methods and a target molecule ("ligand") attached. Potential interacting analytes may be passed over the surface. When an analyte binds, the difference in refractive index between it and the buffer it replaces will lead to a signal which is proportional to the mass of material bound - so large analytes give a bigger signal than small ones. Since the signal (measured in arbitrary Resonance Units) is proportional to mass, a maximum response value (Rmax) can be calculated from the immobilisation level of the ligand and the MW of the ligand and analyte. This allows the experimental results to be assessed in terms of the Rmax value. The practical lower limit for detection is about 400 Da.
If the interaction is reversible, then flowing buffer over the chip will remove the analyte, freeing the surface for another experiment. This may be slow, in which case a "regeneration" reagent e.g. high salt or low pH buffer can be used to encourage dissociation. High affinity interactions on the sensor chip may be difficult to regenerate with either partial regeneration or damage to the sensor surface and many researchers use the Biotin Capture Kit (GE Healthcare) to avoid these complications. The signals are monitored as a function of time and analysis of the association and dissociation phases can provide information on the kinetics of binding (on and off rates) as well as affinity (dissociation constants) from the equilibrium values.
Artefacts due to sample buffer that affect the RI and non-specific binding are removed by simultaneously passing the analyte sample over control and test flow-cells and observing the signal difference between the two.
Due to its widespread use there are many protocols for immobilising commonly used fusion proteins (e.g. His-tagged, GST, biotinylated) and regenerating the surface between samples.
Screening for binding and binding specificity: a target ligand is coated on the surface and a series of different analyte samples passed over - interactions can quickly be seen and ranked in order of strength.
Affinity and kinetics of binding: a series of different analyte concentrations are passed over the ligand and the rates of binding and dissociation are observed. These may be analysed to provide kinetic on and off rate constants. The steady state response to analyte at different concentrations provides a binding isotherm which gives the analyte's affinity for the immobilised ligand. An alternative approach is to apply increasing concentrations of analyte without regeneration and then deconvoluting the resulting curves to obtain the kinetic parameters (single cycle kinetics).
Please speak to the facility manager before preparing reagents so that we can review your experimental approach and make suggestions.
The T200 from Biacore (GE Healthcare) is a research grade instrument for SPR analysis of biomolecular interactions. The system is fully automated with user friendly Wizard based software making it simple to set up experiments which can be run unattended. The analysis software Bia Evaluation is also very easy to use.
The system uses Biacore Series S Sensor Chips - cassettes containing the prepared gold surfaces. These have 4 flow cells allowing two completely independent experiments, or three where one cell can be used as a common reference. Various different surfaces are available - CM5 carboxymethyldextran is the cheapest and is used with the amine coupling procedure to immobilise proteins via surface lysine residues or cysteine residues with the thiol ligand coupling. NTA surface chips are used with His-tagged proteins and streptavidin (SA) chips to immobilise biotin labelled materials e.g. nucleic acids. We carry stocks of the CM5 chips and may have other types that we can supply at cost. The daily charge also includes the standard amine coupling reagents (EDC, NHS and ethanolamine) and we may have other useful reagents and kits that you can try without charge.
Samples & buffers
For typical proteins immobilised as ligands, about 100 µL of material at 10 - 100 µg/ml is required. For the analytes typical injection volumes would be approximately 100 µL with concentration dependent on the required affinity.
Choice of buffers
The ligand immobilisation step requires the sample in a buffer which will not interfere with the process: e.g. for amine coupling this means a low salt low pH buffer free of TRIS and other reactive constituents. Other immobilisation methods may have different constraints.
For the analytes used in the binding association and dissociation phases there is more flexibility: in general the buffer should not compromise activity or stability of the material at the surface. Good matching of the analyte buffer with the background running buffer used is helpful to minimise baseline shifts. This may be achieved by dialysis or dilution.
Regeneration buffers are the most difficult to optimise - they need to abolish interaction so the surface can be fully cleaned of analyte, but without degrading the attached ligand: if these conditions are not met the binding capacity will vary through a series of experiments.
Users prepare their own buffers which should be 0.22 µm filtered and supplemented with 0.05% P20 surfactant. The P20 is intended to prevent protein binding to the pipes and flow channels within the system and its omission may lead to erratic and irreproducible results.
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