Surface Plasmon Resonance Methods and Protocols
While commercial instruments have expanded the usage and the related literature has increased, the quality of surface plasmon resonance (SPR) research has been hindered by a lack of knowledge of the processes that influence the SPR signal. In Surfac
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1. Introduction Biomolecular interactions are at the core of virtually every biological phenomenon: ligand–receptor interactions, signal transduction, regulation of gene expression, etc., are all controlled by specific recognition of interacting partners. So it is not surprising that there is a keen interest in and need for methods and technologies to characterize biomolecular interactions. Such methods and technologies have been developed and improved, especially in recent years. In the broad spectrum of technologies for interaction analysis, label-free biomolecular interaction analysis has a special place. The introduction of labels, e.g., fluorescent labels, N.J. de Mol, M.J.E. Fischer (eds.), Surface Plasmon Resonance, Methods in Molecular Biology 627, DOI 10.1007/978-1-60761-670-2_1, © Springer Science+Business Media, LLC 2010
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to study molecular interactions has brought great progress, but also has drawbacks like the risk to intervene in the binding event. The last decade we have seen tremendous progress in equipment for label-free biosensor techniques, like microcalorimeters, quartz crystal microbalances, reflectometric interference spectrometers, and, not least, surface plasmon resonance (SPR) equipment. SPR has become very important with a yearly impressive increase in number of publications (1). Because it is a label-free technique, only one of the interacting partners has to be immobilized on a sensor surface. SPR has been applied in a wide range of settings, even mimicking biological environments like membranes and surfaces with multiple-binding partners. In the next section, the principles of the SPR phenomenon are described briefly. In essence a mass change near a thin gold (or other metal) surface is detected in real time. This implies that the change in SPR signal over time (as depicted in a sensorgram, see Fig. 1.1) also contains kinetic information. Ideally, the sensorgram contains kinetic information, but also information upon reaching equilibrium (steady state). The signals at equilibrium can be readily used for assay of equilibrium-binding constants (KA or KD , e.g., see Chapter 6). The kinetic phase (association and dissociation) can be used for kinetic analysis, and may yield under proper conditions also equilibrium-binding constants. Kinetic analysis can be easily performed if a simple one-to-one interaction model describes the data, yielding the association rate (kon ) and the dissociation rate (koff ), e.g., see Chapter 2. In practice, often more complex binding models may apply, e.g., due to mass transport (see Section 2 and Chapter 2), heterogeneity of the surface (see Chapter 2), conformational change, multivalent binding, dimer formation. In such cases so-called global kinetic analysis may be performed using specific software like CLAMP or Scrubber (2).
Fig. 1.1. Typical sensorgram of a molecular interaction. The various phases of a SPR experiment are shown. The SPR signal (R) is here expressed as change in SPR angle in millidegree (m◦ ) (see text).
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