BiacoreX100 surface plasmon resonance instrument
Surface plasmon resonance (SPR) studies of protein in the solution phase binding to an immobilised protein surface enables the protein association to be characterized. As the analyte is flowed over the immobilized partner, the response increases, and when buffer is then flowed over the partner, the response decreases. The extent to which different molecules interact with a single partner immobilized on a sensor surface reveals the specificity of an interaction. Apart from the need to immobilise the partner on a chip surface, the method is artefact-free and does not require the use of any labelling.
Kinetic measurements involve the on-rate and off-rate and their ratio gives the dissociation constant KD values. If the on-rate and off-rate are rapid, only the overall response is measured in what are termed equilibrium measurements. The interaction stoichiometry is measured from comparing the increase in response units to the amount of response units immobilised on the sensor surface.
Farfield dual polarisation interferometer
Macromolecules can change shape when immobilised on a sensor surface and then subsequently treated with different buffers, small ligands or other macromolecules. Unlike surface plasmon resonance, these shape changes are measurable using a dual polarisation interferometer. This approach can often provide vital clues on bimolecular mechanisms, especially when considering interactions involving proteins immobilised on cell surfaces. Like surface plasmon resonance, information on dissociation constants is also obtained.