Fluorescence polarization must be classified as a unique detection method relative to other intensity-based methods because both bound and unbound tracer is measured. Other fluorescence techniques require either physical removal of unbound tracer or a means for distinguishing only bound tracer. The presence of unbound tracer in the detection volume has profound consequences on how assay performance is gauged. The intent here is to provide tools for accurate assay performance assessment and to discuss some of the practical considerations necessary for understanding the advantages and limitations of the technology. An emphasis is placed on applications using G protein-coupled receptors.
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