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Jan 15, 2009 (Vol. 29, No. 2)

Optimization of Binding Interaction Studies

New Approach Advocates Interspot Referencing Using Multiplexed SPR Arrays

  • Surface plasmon resonance (SPR) has revolutionized the study of the intricate protein interactions that are required for the execution and maintenance of complex biological processes, enabling the elucidation of the roles that intracellular concentration, ionic environment, cofactors, and protein conformation play in maintaining those processes. SPR is also a powerful tool for the rapid identification of highly specific monoclonal antibodies with high affinity for the analyte of interest, detailed evaluation of drug lead compounds, and optimization of affinity protein purification methods.

    Surface plasmon resonance optical biosensing requires neither radiochemical nor fluorescent labels to provide real-time data on the affinity, specificity, and kinetics of protein interactions, and it can detect picomolar levels of proteins and other molecules as small as a few hundred daltons. SPR occurs when light interacts with a metal film placed at the interface between two media with different refractive indices, such as glass and water.

    SPR biosensors respond in real time to changes in the refractive index at the interface resulting from the binding and subsequent separation of two molecules, one of which is bound to the sensor surface. As the molecule in solution (analyte) flowing over the surface binds to the immobilized molecule (ligand), the refractive index near the sensor surface increases, leading to a shift in the SPR angle. When the complex on the sensor surface dissociates, the SPR angle shifts back.

    These changes in refractive index, expressed in response units (RU), are proportional to mass changes at the surface of the sensor chip. Optimized association, dissociation, and equilibrium constants can be calculated by fitting response data for a range of analyte concentrations under identical reaction conditions to a computational model.



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