No method for quantifying molecular interactions is foolproof, and FAC is no exception. The primary concern with FAC relates to assay development - it can take considerable time and effort to design the assay, in which a protein or some other appropriate biomolecule requires immobilization. The classical concern with immobilization-dependent assays involves preservation of relevant activity; it is not uncommon for initial attempts to be completely unsuccessful especially when chemical labelling strategies are used (e.g. Schiff base reactions between a protein and an amino-resin). More common is the generation of a partially deactivated protein stationary phase, which begs the question whether the loss of activity is simply a ''percentage problem'' or reflective of an alteration of protein structure/dynamics. Most often this can be resolved by comparing the results generated by FAC analysis of a known ligand, to that generated by an independent assay. Obviously this requires some idea of what structural features constitutes a ligand. This represents the key restriction to extending this assay type to true ''orphan receptors'' or newly discovered target molecules: this knowledge may not be readily available. It is almost always true that a successful immobilization/retention strategy can be developed and in some cases the immobilized form is much closer to in vivo conditions than true homogeneous assays (consider membrane-bound receptors for example).
The FAC method offers the opportunity to measure binding events in an environment of undistorted equilibrium, which is a strength but also an analytical challenge. There is no inherent purification or enrichment, and as a result the chosen detector must meet some stringent performance requirements. Systems have used simple UV-based flow cell detectors, which is entirely appropriate for very simple single-compound analyses. The FAC-FD system of Hirabayashi incorporates fluorescence detection of labelled marker ligands, as a sensitive and selective means of quantifying an interaction and is particularly useful in higher screening-rate single-compound analyses, where the fluorescently labelled ligand is used competitively . Both approaches preserve the essence of the FAC advantage (accurate/precise measurements, access to a wide range of binding strengths), but both need a high degree of purity in the samples to be analyzed. This is required for most plate-based or biosensor-based assay systems, and does not provide significant justification for developing a FAC assay, aside from validating the results of other methods or exploring very weak interactions.
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