Protein interaction with fluorescent compounds

Fluorescence spectroscopy offers several inherent advantages for the characterization of molecular interactions and reactions. Firstly, it is 100-1000 times more sensitive than other spectrophotometric techniques. Secondly, fluorescent compounds are extremely sensitive to their environment. For example, vitamin A that is buried in the hydrophobic interior of a fat globule has fluorescent properties different from molecules that are in an aqueous solution. This environmental sensitivity enables characterization of viscosity changes such as those attributable to the thermal modifications of triglyceride structure, as well as the interactions of vitamin A with proteins. Third, most fluorescence methods are relatively rapid (less than 1 s with a Charge Coupled Device detector). One particularly advantageous property of fluorescence is that one can actually see it since it involves the emission of photons. The technique is suitable for at-line and on/in-line process control. 

The high FRET efficiency obtained in HTRF assays enables a large variety of biological events to be probed. Protein/protein interactions, enzymatic activities (e.g. kinases or proteases), or a large number of biomarkers have been detected with a very low detection limit. Like the other homogeneous fluorescent technologies like FP or FCS, HTRF can be easily automated and miniaturized down to a 1536 weUs plate format for the HTS of large libraries of chemical compounds. ... 

Microarrays [287] are matrices with spots of different chemical compounds on a surface (Fig. 18a). The number of spots ranges from a few dozen to up to several millions. The microarray is incubated with the sample and each spot interacts with the sample in parallel, leading to as many parallel assays as there are spots on the microarray. T q)ically a microarray is read out by fluorescence and used for nucleic acid or protein analysis. 

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