At an interface between two transparent media of different refractive index (glass and water), light coming from the side of higher refractive index is partly reflected and partly refracted.
Above a certain critical angle of incidence, no light is refracted across the interface, and total internal reflection is observed. While incident light is totally reflected the electromagnetic field component penetrates a short (tens of nanometers) distance into a medium of a lower refractive index creating an exponentially detenuating evanescent wave. If the interface between the media is coated with a thin layer of metal (gold), and light is monochromatic and p-polarized, the intensity of the reflected light is reduced at a specific incident angle producing a sharp shadow (called surface plasmon resonance) due to the resonance energy transfer between evanescent wave and surface plasmons.
The resonance conditions are influenced by the material adsorbed onto the thin metal film. Satisfactory linear relationship is found between resonance energy and mass concentration of biochemically relevant molecules such as proteins, sugars and DNA. The SPR signal which is expressed in resonance units is therefore a measure of mass concentration at the sensor chip surface. This means that the analyte and ligand association and dissociation can be observed and ultimately rate constants as well as equilibrium constants can be calculated.
SPR-based instruments use an optical method to measure the refractive index near (within ~300 nm) a sensor surface.
•One molecule (the ligand) is immobilised onto the sensor surface.
•Its binding partner (the analyte) is injected in aqueous solution (sample buffer) through the flow cell, also under continuous flow.
•As the analyte binds to the ligand the accumulation of protein on the surface results in an increase in the refractive index.
•This change in refractive index is measured in real time, and the result plotted as response or resonance units (RUs) versus time (a sensorgram).
•Background response must be minimized by making blank run.this data will be subtracted from the real data.
What SPR is good for
we can show that the recombinant protein has the same structure as its native counterpart.
we can confirm that the protein binds its natural ligands.
Because such interactions involve multiple residues, which are usually far apart in the primary amino acid sequence, they require a correctly folded protein.
real-time binding data
obtaining accurate kinetic data is a very demanding and time-consuming task, and requires a thorough understanding of binding kinetics and the potential sources of artefact
mass transport limitations kon values faster than about 106 M-1s-1.
koff values slower than 10-5 s-1 or faster than ~1 s-1 is difficult.
Analysis of mutant proteins
capture of proteins from crude mixtures onto the sensor surfacefor analysing mutants generated by site-directed mutagenesis.
Mutants can be expressed as tagged proteins by transient transfection and then captured from crude tissue culture supernatant using an antibody to the tag
Evaluating effect of mutation on binding properties
quantifying the effect of mutations on the thermodynamics and kinetics of weak protein/ligand interactions