Fluorescein isothiocyanate, fluorescence

Calibration. Many approaches have been used to calibrate flow cytometric measurements. Including the comparison of flow and nonflow techniques (radiolabels, spectrofluorometry). In recent years, commercial standards have been introduced which are calibrated in fluorescein equivalents/particle (e.g., 3,000 or 500,000). With labeled ligands, calibration requires determining the relative quantum yield of the ligand compared to pure fluorescein and using the standards to analyze the amount bound on cells. Our ligands (fluorescein isothiocyanate derivatives) are typically 50% as fluorescent as fluorescein. 

Labelling Na,K-ATPase with ATP analogues provides evidence for contribution from charged residues that are widely separated in the sequence of a subunit of Na,K-ATPase. The first indication came from ATP sensitive covalent insertion of fluorescein-isothiocyanate (FITC) into Lys ° in the a subunit [90], The strong fluorescence signal provides a convenient probe for monitoring conformational transitions in the proteins. Site-directed mutagenesis of Lys reduces the activity of... 

The nondestructive introduction of a fluorescent label would provide the molecule with a nonradioactive fluorophore, yet would preserve the option for direct radiolabelling of the fluorescent moiety with 125Iodine. This approach was pioneered by Nagasawa et al. (5) who reacted native or /V-desulfated heparins with a fluorescein isothiocyanate (FITC). The resulting degree of labelling was low... 

Muramoto K, Nokihara K, Ueda A, Kamiya H (1994) Gas-phase microsequencing of peptides and proteins with a fluorescent Edman-type reagent, fluorescein isothiocyanate. Biosci Biotechnol Biochem 58 300-304... 

Wu S, Dovichi NJ (1989) High-sensitivity fluorescence detector for fluorescein isothiocyanate derivatives of amino acids separated by capillary zone electrophoresis. J Chromatogr 480 141-155... 

Fluorescein isothiocyanate (FITC) is one of the most popular fluorescent probes ever created. An isothiocyanate derivative of fluorescein is synthesized by modification of its lower ring at... 

In addition to the wide range of commercial probes, many other fluorescent molecules have been synthesized and described in the literature. Only a handful, however, are generally used to label antibody molecules. Perhaps the most common fluorescent tags with application to immunoglobulin assays are reflected in the main derivatives produced by the prominent antibody manufacturing companies. These include derivatives of cyanine dyes, fluorescein, rhod-amine, Texas red, aminomethylcoumarin (AMCA), and phycoerythrin. Figure 20.16 shows the reaction of fluorescein isothiocyanate (FITC), one of the most common fluorescent probes, with an antibody molecule. 

Figure 5 shows two typical core-shell structures (a) contains a metal core and a dye doped silica shell [30, 32, 33, 78-85] and (b) has a dye doped silica core and a metal shell [31, 34]. There is a spacer between the core and the shell to maintain the distance between the fluorophores and the metal to avoid fluorescence quenching [30, 32, 33, 78-80, 83]. Usually, the spacer is a silica layer in this type of nanostructures. Various Ag and Au nanomaterials in different shapes have been used for fluorescence enhancement. Occasionally, Pt and Au-Ag alloys are selected as the metal. A few fluorophores have been studied in these two core-shell structures including Cy3 [30], cascade yellow [78], carboxyfluorescein [78], Ru(bpy)32+ [31, 34], R6G [34], fluorescein isothiocyanate [79], Rhodamine 800 [32, 33], Alexa Fluor 647 [32], NIR 797 [82], dansylamide [84], oxazin 725 [85], and Eu3+ complexes [33, 83]. 

Figure 5 Immunopotentiating reconstituted influenza virosomes (IRIV) adjuvance on cytotoxic T-cell (CTL) induction. PBMC from a healthy donor were cultured in the presence of influenza matrix (IM)58 66 (A), IMss-eo and control liposomes (B) or IMss-ee and IRIV (C). After a seven-day culture, percentages of IMss-ee speciflc CTL within cultured cells were quantifled by HLA-A0201/IM58 gfi phosphatidylethanolamine tetramer staining (fluorescence 2) and anti CDS fluorescein isothiocyanate staining (fluorescence 1). CTL precursor frequencies detected in IMss-ee and IRIV stimulated cultures within the same experiment are shown in (D). Source From Ref 6.

Extending the utility of fluorescence to various nonfluorophores is achieved via chemical derivatization methods, also termed labeling or tagging methods (Reaction 11.2). Numerous commercial fluorescent tags are available with disparate reactive functional groups. For example, derivatives of fluorescein, fluorescein isothiocyanate (FITC) are reactive toward nucleophiles such as amines and sulfhydryl groups. 

A fluorescent tag (fluorescein isothiocyanate) attached to the lectin Concanavalin A is useful in staining yeast cell wall fragments (Siebert et al., 1981). Concanavalin A specifically binds to mannan, which is prominent in yeast cell walls. 

The dissociation constant (Kd) of a monoclonal antibody with fluorescein isothiocyanate- (FITC)-labeled insulin and unlabeled insulins from several species were measured using CE with laser-induced fluorescence detection (CE-LIF) (9). Kd determinations were made by separating free FITC-labeled insulin and its complex with the antibody in equilibrated solutions in 6 s or less (Fig. 3). Dissociation and association rates for insulin, FITC-insulin, and the antibody are fast enough to reach equilibria in less... 

Add 33 pi of fluorescein isothiocyanate (FITC tetramefliylrho-damine isothiocyanate, TRITC, or another fluorescent dye isothiocyanate derivative is used the same way), 50 mg/ml in DMF, to 1 ml of 5 mg/ml IgG in Soln. A. Shake at RT protected from light for 1 h. Remove surplus FITC and its hydrolysis products on a Sephadex G-25 column, equilibrated with PBS. The conjugate appears in the void volume and should be concentrated by ultrafiltration. Add sodium azide to a final concentration of 0.02% (w/v) and glycerol up to 10% (w/v) and store at 4 °C. 

Fluorescein isothiocyanate, a fluorescent label often employed in biochemistry. 

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