Fluorescence displacement assay

Kinkaid, A. and Wilton, D.C. 1991. Comparison of the catalytic properties of phospholipase A2 from pancreas and venom using a continuous fluorescence displacement assay. Biochem. J. 278, 843-848. 

Anslyn showed by variable temperature NMR that the homoazacalix[4]arene 90 at room temperature had several conformations, with the more stable cone conformation freezing out at —70 °C. Anionic guests were shown to bind to 90 by a fluorescence displacement assay [58]. 

There are several other techniques Uke the fluorescent dye displacement assays, footprinting, Fourier transform infrared spectroscopy. X-ray crystallography, electron microscopy, confocal microscopy, atomic force microscopy, surface plasmon resonance etc used for hgand-DNA interactions that are not discussed here. 

Indicator displacement assays (IDAs) - or, in the specific case of fluorescent indicators, F-IDAs - are based on the next alternative concept described here. A receptor with an affinity for a given analyte is loaded with an indicator, usually a fluorescent or colored dye, whose spectral properties undergo a change upon complexation with the receptor. Treatment of this indicator-receptor complex with the analyte results in the displacement of the indicator from the receptor and a restoration of the indicator s original spectral properties, indirectly reporting analyte coordination (Fig. 27). For effective detection, two main requirements have to be fulfilled (1) the receptor/indicator interaction must be reversible and weaker than the interaction of the receptor with the designated analyte and (2) the indicator must show significantly different optical properties when bound to the receptor and when freely dissolved in solution. 

Fig. 27 (a) Representative scheme for a displacement assay protocol in which first a fluorescent indicator is coordinated to a host. As a consequence, its optical properties are altered Second upon analyte addition, the higher affinity of the analyte for the host leads to dissociation of the complex and displacement of the indicator. The original optical properties of the fluorophore are restored, signaling indirectly the presence of the analyte (b) Some examples of receptors and fluorescent indicators reported in the literature for F-IDAs... 

Horie S, Kubo Y (2009) Fluorescence-based indicator displacement assay for phospho-sugar detection using zinc(II) dipicolylamine-appended phenylboronic acid. Chem Lett 38 616-617... 

Glutathione S transferases bind bile acids in vitro but doubt has been cast over whether this happens in vivo as these enzymes were not labelled by fluorescently labelled bile acids in experiments to identify the carrier proteins but may play a role with the raised levels in cholestasis. Liver fatty-acid-binding protein has been shown to bind bile acids by using a displacement assay with fluorescent fatty-acid ligand. This work clearly showed displacement to be directly related to hydrophobicity, such that lithocholate conjugates had the greatest effect. This may indicate a mechanism to minimise toxicity within the hepatocyte. 

Scheme 11.7 Fluorescent indicator displacement assay for acetylcholine developed by Inouye in 1994.31...

The Anslyn group has pioneered supramolecular displacement assays as a distinctive method of analysis. A typical example used a tripodal molecule, or podand, to determine the amount of citrate in drinks and the quality of wine made from Pinot Noir grapes [7], The initial work on citrate utilized a complex between a tris(guanidinium) derivative of 1,3,5-triethylbenzene with a fluorescent dye, car-boxyfluorescein. In the presence of citrate the dye is ejected and replaced with the... 

Fig. 16 Ethidium bromide displacement assay of the DMAEC-a 18-SS-polyrotaxanes polyplex and the LPEI22k polyplex. The recorded fluorescent intensities (F.I.) were expressed relative to the fluorescence intensity of the DNA-EtBr solution in the absence of polycation, after subtracting the fluorescence of EtBr in the absence of DNA under the same buffer conditions...

A fluorescent intercalator displacement assay for establishing DNA-binding selectivity and affinity 04ACR61. 

Competition-in-solution assays can quantify the effect of library components on the interaction between components of a protein complex. These assays include traditional biochemical assays that measure interactions through antibody- or tag-mediated pull-down or label displacement assays, along with biophysical methods that measure changes in the property of a component upon binding to its partner protein, such as fluorescence spectroscopic assays and SPR. It is essential for assay development that the requirements (affinity and binding site) for assembly of the interaction partners be at least partially defined such that a tractable model system for the target interaction can be developed, as was the case for MDM2/p53, IL-2/IL2-aR and Bcl/Bak [138-140],... 

Among the first modern indicator displacement assays in supramolecular chemistry was the fluorescence-based detection of the neurotransmitter acetylcholine developed by Inouye, Scheme 11.7.31 The fluorescence of the pyrene derivative is quenched by binding to a deprotonated... 

Reversible dye binding systems can be developed into colourimetric and fluorescent assays for analytical detection. As shown in Figure 11.3, two different binding schemes can be envisioned, (a) competitive dye/analyte inclusion, and (b) cooperative dye/analyte inclusion. The competitive inclusion process is the basis for the dye displacement assay where the analyte displaces the dye from the container molecule. For this process to be visualized, the properties of complexed and uncomplexed dyes must be markedly dif-... 

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