Fluorescein Isothiocyanate,

The most used EIA reagents conjugate a fluotophote such as fluorescein-isothiocyanate (EITC) or thodarnine—isothiocyanate to antibody (or antigen) free amino groups. Examples of other commonly used fluotophotes for EIA and their spectral characteristics ate presented in Table 3. EIA assays ate available in sandwich and competitive formats similar to EIAs. Unlike EIA kits which can be used directly with visual color deterrnination, EIAs require a fluorometer, and thus ate primarily laboratory-based. 

Figure 9.8 Schematic illustration of the two-dimensional HPLC/fast-CZE instmmental setup. The argon laser beam was set at 488 nm, which was used to photodegrade and detect the fluorescein isothiocyanate tag.

The transfection mechanism of plasmid-chitosan complexes as well as the relationship between transfection activity and cell uptake was analyzed by using fluorescein isothiocyanate-labeled plasmid and Texas-Red-labeled chitosan. Several factors affect transfection activity and cell uptake, for example the molecular mass of chitosan, stoichiometry of complex, seriun concentration and the pH of the transfection medium. The level of transfection with plasmid-chitosan complexes was found to be highest when the molecular mass of chitosan was 40 or 84 kDa, the ratio of chitosan nitrogen to DNA phosphate was 5, and serum at pH 7.0 was 10%. Plasmid-chitosan complexes most likely condense to form large aggregates (5-8 p,m), which absorb to the cell surface. After this, plasmid-chitosan complexes are endocytosed, and accumulate in the nucleus [97]. 

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. 

Spray solution 1 Dissolve 10 mg fluorescein isothiocyanate in 100 ml 0.1 mol sodium hydroxide solution. 

The amino acids probably react with the fluorescein isothiocyanate to yield fluorescein thiourea derivatives. These are hydrolyzed at elevated temperature in alkaline medium so that the amino groups that are produced can then react with ninhydrin. 

Tible 1 Color reactions of amino acids with fluorescein isothiocyanate — ninhydrin (extract from original table). 

FIG. 9 Confocal laser scanning micrograph of a hollow polymer capsule. The polymer capsule was obtained from polymer multilayer-templated FDA microcrystals after removal of the colloidal core. The FDA microcrystals were coated with SDS and 11 polyelectrolyte layers [(PAH/PSS)3/PAH/ (PSS/PAH-FITC)2]. (PAH-FITC = PAH labeled with fluorescein isothiocyanate.) The microcrystal core was removed by exposure of the coated microcrystals to ethanol, causing solubilization of FDA. 

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... 

Fe-TPAA Fe(III)-tris[N-(2-pyridylmethyl)-2-aminoethyl] amine Fe-TPEN Fe(II)-tetrakis-N,N,N, N -(2-pyridyl methyl-2-aminoethyl)amine FFA Free fatty acids FGF Fibroblast growth factor FID Flame ionization detector FITC Fluorescein isothiocyanate FKBP FK506-binding protein FLAP 5-lipoxygenase-activating protein... 

Miniaturized columns have provided a decisive advantage in speed. Uracil, phenol, and benzyl alcohol were separated in 20 seconds by CEC in an 18 mm column with a propyl reversed phase.29 A19 cm electrophoretic channel was etched into a glass wafer, filled with a y-cyclodextrin buffer, and used to resolve chiral amino acids from a meteorite in 4 minutes.30 A 6 cm channel equipped with a syringe pump to automate sample derivatization was used to separate amino acids modified with fluorescein isothiocyanate.31 Nanovials have been used to perform tryptic digests on the 15 nL scale for subsequent separation on capillary Electrophoresis.32 A microcolumn has also been used to generate fractions representing time-points of digestion from a 40 pL sample.33 A disposable nanoelectrospray emitter has been... 

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... 

This online instrumental setup was applied for the 2D separation of fluorescein isothiocyanate-derivatized human urine the results are shown in Fig. 16.7. This separation used overlapped CZE runs, which means that two samples are injected on the column, one after the other, separated by a period of time that will allow the separation of the first-injection components from the second-injection components that are present in the capillary at any point prior to detection. This was accomplished by making an injection in a time interval of exactly half of the actual CZE run time. To overlap the CZE runs, an injection was made every 29 s. For the overlapping to work,... 

Among the commercially available fluorescein derivatives that have been widely used are fluorescein isothiocyanate (FITC) 49, carboxyfluorescein (FAM) succini-midyl ester 50, and fluorescein dichlorotriazine (DTAF) 51. FITCs are the most commonly used fluorescein derivatives. They have been used to react with sulfhy-dryl [127], targeting reduced cysteine chains and especially amino groups in peptides or proteins [128]. 

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... 

Houston B, Peddie D (1989) A method for detecting proteins immobilized on nitrocellulose membranes by in situ derivatization with fluorescein isothiocyanate. Anal Biochem 177 263-267... 

Jackson RJ, Mendlein J, Sachs G (1983) Interaction of fluorescein isothiocyanate with the (H+ + K+)-ATPase. Biochim Biophys Acta 731 9-15... 

Fig. 20.1. Confocal images of whole mounts of the ovijector region of A suum stained with phalloidin-tetramethylrhodamine isothiocyanate (TRITC) to show muscle and with an anti-RFamide antiserum coupled to fluorescein isothiocyanate (FITC) to show FaRPergic nerves. (A) Main ventral nerve cord encircles opening of ovijector where it meets the body wall and is immunopositive for FaRPs. (B) Flat-fixed preparation of the ovijector showing circular muscles and tracts of parallel FaRPergic nerves (arrows). (C) Detail of the circular muscle of ovijector and associated nerves (arrows). (D) A FaRPergic cell body is localized in the ventral nerve cord at junction with ovijector and provides innervation to ovijector muscle.

To be used as delivery carriers for biomolecules, first it is essential to check whether the conjugates can effectively enter cells. Cellular uptake experiments were performed by using fluorescein isothiocyanate (FITC) conjugated LDH as a probe. Cells (5 x 105/ 1 ml) were incubated with LDH-FITC and its uptake was measured by flow cytometry. As shown in Figure 13.4, the cellular uptake was time and concentration dependent and... 

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