Streptavidin-fluorophore

Detection Using a Biotin-Streptavidin-Fluorophore System... 

Apply a solution containing the streptavidin-fluorophore to the sections for 30 min (see Note 44). 

Wash the sections with PBS for 15 min to remove unbound streptavidin-fluorophore (see Note 43). 

Streptavidin is available linked to fluorophores, such as Texas red It is usually purchased as a solution that can be used at a dilution of approx 1.200 in PBS-BSA. The solution containing the streptavidin-fluorophore conjugate is applied to the sections for 30 min Thereafter, sections are washed and treated as if they had been labeled with a fluorescently tagged secondary antibody... 

Although biotin-fluorophores have been described in the detection of biomolecules, namely proteins, peptides, or DNA, most of these loose part of their fluorescence when binding to the tetrameric proteins avidin and streptavidin. 

Agiamamioti and co-workers [72] synthesized a novel biotinylated fluorophore, 10-(2-biotinyloxyethyl)-9-acridone 25 with favorable properties for bioanalytical applications. In aqueous solutions, it displayed high fluorescence ([Pg.37]

First entry on each line is the tag. Square brackets enclose the probe donor + transfer enzyme (where applicable) or reaction. Probe organic fluorophores, nanoparticles (can be QDs as in methods 2, 3, 8, 9) or a bridging/recognition moiety. If desired, the latter can serve in a second orthogonal reaction ( piggyback strategy). See text. Biotin readout probes linked to avidin, streptavidin, anti-biotin 7[101] ... 

Make it visible the fluorophore label can be visualized directly using fluorescent microscopy. The biotin label (see Sect. 6.2.1) can be detected using streptavidin conjugated with an enzyme the latter must be visualized through an enzyme chromogenic system. Incubate sections with an appropriate enzyme substrate until optimal color develops (see Sect. 2.3). 

Fig. 5.2. Methods for detection of passenger-ligand interaction. (A) Fluorophore-coupled ligand (B) fluorophore-coupled antibody (C) quaternary complex generated by subsequent rounds of incubation with ligand, primary antibody, biotinylated second antibody, and strep ta-vidin, R-phycoerythrin conjugate (D) quaternary complex generated by subsequent rounds of incubation wi tli ligand, primary antibody, biotinylated second antibody, and streptavidin-coated magnetobeads. L ligand P passenger.

The presence of biotin labels on an antibody molecule provides multiple sites for the binding of avidin or streptavidin. If the biotin binding protein is in turn labeled with an enzyme, fluorophore, etc., then a very sensitive detection system is created. The potential for more than one labeled avidin to become attached to each antibody through its multiple biotinylation sites provides an increase in detectability over antibodies directly labeled with a detectable tag. 

The following sections present suggested protocols for labeling avidin or streptavidin with selected fluorophores. Other fluorescent probes may be constructed using the reagents and methods discussed in Chapter 8, Section 1. 

In a darkened lab and with gentle mixing, slowly add 50—100 p,l of the fluorophore solution to each milliliter of the avidin or streptavidin solution. 

Modification of avidin or streptavidin with Texas Red sulfonyl chloride may be done similarly, except the fluorophore is first dissolved in acetonitrile prior to addition to the aqueous reaction mixture. 

The concept of MW enhancers was developed to increase polarization value changes caused by large size differences between substrate and product when small peptide substrates were used. The most frequently used approach is based on the attachment of a biotin molecule to the terminus of the peptide on the opposite site of the scissile bond to the fluorophore. After the incubation of the substrate with the protease, streptavidin is added to the assay. Streptavidin binds to biotin and enhances the masses of the substrate and the unlabeled product, thus causing an increase of the polarization... 

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