Surface Plasmon Resonance is used for monitoring and quantification of interaction of biomolecules and requires one of the interacting partners (the ligand) to be immobilized on a functionalized surface, the so-called SRP chip. SPR measures the reflections of light on the backside of the chip, in particular the angle of the part of the reflected light that is absorbed due to the plasmon resonance wave propagating near the immobilized ligands. This angle is very sensitive to changes on the chip, such as adsorption via binding of an analyte to the ligand. Both the association (ka) and dissociation (kd) rates are measured yielding the dissociation constant KD. Typical KD ranges measureable are (less than) nM up to mM.
What are the sample requirements?
- Pure and homogeneous samples are required. This is absolutely critical for the amine-coupling method and for all analytes to be tested.
- About 10-50 ug in 200 uL, i.e. 50-250 ug/ml, of a protein is needed for ligand immobilization on the sensor chip.
- About 200-300 ul of the analyte is needed at 10x expected Kd.
- For best results a 100-fold range of analyte concentrations should be attempted at 0.1–10 x Kd
What other specific considerations are relevant?
- Concentration of the analyte needs to be determined as accurately as possible.
- Running buffers: Most commonly used buffers are HBS-EP, TBS-P and PBS-P.
- Immobilization buffer: The acetate buffer (at pH 4.0, 4.5, 5.0, or 5.5) is often recommended
- Running buffer and analyte buffers should be matched
- Analytes need to be larger than 150 Da
Partners offering this technique
MOSBRI reference partner sites for this technique: