FITC . See Fluorescein

Ferromagnetic-paramagnetic transition temperature, 395,396 Ferromagnetism, 433 FITC. See Fluorescein isothiocyanate Flame, 294, 295, 298, 312 assisted spray pyrolysis, 388... 

Fluorescein-labeled proteins are obtained by reacting proteins with FITC (see Fig. 3). Dissolve the protein (small proteins 0.1-0.3 mg, large proteins or antibodies 0.1-1 mg)in 100 pi of 0.1 M sodium phosphate buffer (pH 8.0). If protein activity is decreased by phosphate ions, the labeling reaction can be carried out in 0.1 M bicarbonate, HEPES, or borate buffers at the same pH. However, do not use buffers containing free amino groups such as Tris or glycine these buffers will react with FITC. 

Although FITC and other reactive fluorescein derivatives still are widely used to label (strept)avidin and other proteins, better fluorescence yield and stability will be obtained if one of the newer hydrophilic fluorescein dyes is used. See Chapter 9, Section 1, for additional details on labeling proteins with fluorescein. 

Albert H. Coons was the first to attach a fluorescent dye (fluorescein isocyanate) to an antibody and to use this antibody to localize its respective antigen in a tissue section. Fluorescein, one of the most popular fluorochromes ever designed, has enjoyed extensive application in immunofluorescence labeling. For many years, classical fluorescent probes such as FITC or Texas red (TR) have been successfully utilized in fluorescence microscopy. In recent decades, brighter and more stable fluorochromes have continually been developed (see Table 14.1). Marketed by a number of distributors, cyanine dyes, Cy2, Cy3, Cy5, Cy7, feature enhanced water solubility and photostability as well as a higher fluorescence emission intensity as compared to many of the traditional dyes, such as FITC or TR. 

The quantification of fluorescent particles in cellular systems is difficult because several aspects such as autofluorescence, bleaching (see below), and quenching hamper analysis. Keep in mind that many fluorophores show a pH-dependent change in emission spectrum and intensity fluorescein-labeled dextrans (FITC-dextran) and calcein are strongly quenched upon acidification. If available, one should read the fluorescence intensity at its isosbestic point, where the intensity is not pH dependent. 

Chapter 5). Separation of a mixture of BODIPY and fluorescein was achieved in less than 20 s (see Figure 6.23). Five bioactive peptides (papain inhibitors, proctobin, opioid fragment 90-95, ileangiotensin HI, and angiotensin III) were also separated in this manner [639]. A similar method has been used to separate two coumarin dyes or to separate Alexafluor-labeled angiotensin from the excess dye [587,640]. CEC separation of four FITC-labeled synthetic peptides was... 

Surface-exposed parts of the protein have been distinguished by their ability to bind specific antibodies and proteolytic enzymes under nondenaturing conditions (see Figure 16). Many of these epitopes are located at positions in the sequence that correspond to the external side of the suggested nucleotide-binding fold (Mate et al., 1992). Antibodies to fluorescein bind only to denatured FITC-labeled Ca2+-ATPase, and not to the native FITC-labeled enzyme. This is consistent with a location of bound FITC in a hydrophobic cleft corresponding to the ATP site. 

Certain fluorescent compounds, such as formycin nucleotides and eosin, behave as noncovalently binding ATP analogs, and their fluorescence increases upon association with the ATPases. The transition from the Na+-form to the K+-form of Na+-K+-ATPase gives rise to a decrease in fluorescence from these probes. The fluorescence of fluorescein isothiocyanate (FITC) bound covalently at the nucleotide site of Na+-K+-ATPase (Table 3) also decreases in relation to transition from the Na+-form to the K+-form. With the noncovalent as well as with the covalently-binding probes the fluorescence decrease probably reflects the structural change in the nucleotide site associated with the reduced nucleotide affinity in the E2 form (see above). By contrast, when FITC is attached to the SR Ca2+-ATPase (at the residue homologous to the FITC-binding residue in Na+-K+-ATPase) the fluorescence increases upon removal of Ca2+. Most likely this relates to the above discussed difference between Na+-K+-ATPase and Ca2+-ATPase with respect to the existence of a stable E2 form with low affinity for nucleotide. 

Cells in the appropriate medium for this example, isolated mouse spleen cells washed and resuspended in PBS at approx 1 x 10 cells/mL. Fluorescein isothiocyanate (FITC)-conJugated goat antimouse immunoglobulin diluted 1 20-1 100 in PBS (see Note 1). 

Not many compounds fluoresce naturally. However, some compounds, when added to another compound, cause that compound to fluoresce. These compounds are called fluorophores. Dansyl chloride and fluorescamine are two that are used to react with primary amines. o-Phthalaldehyde is used as a postcolumn reactant to produce fluorescent compounds. Other compounds are naphthalenedialdehyde (NDA), fluorescein isothiocyanate (FITC), and phenylthiohydantoin (PTH). See Chapter 19, p. 203, for more detail. 

The observed release pattern from both types of microspheres lies in the distribution of the protein inside a microsphere, which is associated with the preparation method. In order to see this, FITC (fluorescein isothiocyanate)-insulin incorporated microspheres were observed under a confocal microscope, as shown in Fig. 7. For Msp A, a homogeneous distribution of fluorescence was observed while Msp B exhibited a rather heterogeneous distribution of FITC-insulin. In addition, Msp B shows significant surface fluorescence. These observations are, hence, consistent with the observed initial burst from Msp B and from the constant insulin release from Msp A over a prolonged period of time. It is reported that the constant release of insulin from triblock copolymer hydrogel may be attributed to the hydrophilic/hydrophobic domain structure of the gel. The incorporation of a significant fraction of insulin in the hydrophobic domain may have made possible... 

Fluorescein isothiocyanate (FITC) and dansyl-chloride were among the first extrinsic fluorescent labels for proteins used for immunofluorescence microscopy and polarization measurements. Fluorescein and rho-damine labels were extensively employed due to their bright emission in the visible range. These probes have drawbacks including hydrophobicity, small Stokes shifts, and sensitivity to pH and photobleaching, which led to the development of new dyes such as Alexa, Bodipy, and cyanine dye families (see Table 1). These dyes cover a broad... 

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