Labeling with TRITC

The level of TRITC modification in a macromolecule can be determined by measuring its absorbance at or near its characteristic absorption maximum ( 575nm). The number of fluor-ochrome molecules per molecule of protein is known as the F/P ratio. This value should be measured for all derivatives prepared with fluorescent tags. The ratio is especially important in predicting the behavior of antibodies labeled with TRITC. For a TRITC-labeled protein, the ratio of its absorbance at 575-280 nm should be between 0.3 and 0.7. 

Fluorescence detection relies on the visualization of a secondary antibody that has been labeled with a fluorophore such as fluorescein (FITC), Texas Red, Tetramethyl rhodamine (TRITC), or R-phycoerythrin. Although this method of detection has a reduced sensitivity of twofold to fourfold compared to chemiluminescence detection, it presents a tenfold greater linear dynamic range, thus providing better linearity and better quantiflcation within the detection limits. Since secondary antibodies can be labeled with fluor-ophores of distinct colors, multiplexing (simultaneous detection of several antigens) of the same blot is feasible. 

Figure 11.4 Elimination of plasmid DNA from the cytoplasm of microinjected cells. Double stranded fluorescein labeled pGL2 plasmid (0.1 pg/ml) was co-injected with TRITC-dextran into the cytosol of HeLa cells and incubated under tissue culture conditions for the indicated time. Cells were fixed and fluorescence micrographs were taken to visualize the distribution of TRITC-dextran (right column) and fluorescein-labeled pGL2 (left column) of the same field, (see Color Plate 11)...

Finally, a double labelling with antibodies and a viability substrate can be performed. De Vos and Nelis (2003, 2006) combined ChemChrome V6 with tetram-ethyl rhodamin isothiocyanate (TRITC) labelled antibodies for the detection of Aspergillus fumigatus. In these approaches, the ChemChrome reagent, yielding green fluorescence, ensures the primary detection by the ChemScan, whereas the TRITC label results in red fluorescence, to be observed microscopically. 

To illustrate the FRET-based glucose assay, we describe the results using a ConA labeled with 7-amino-4-methyl-coumarin (donor) and a-D-Mannoside labeled with tetramethylrhodamine cadaverine (TRITC-cadaverine) as the acceptor (Figure 6) (65). This donor-acceptor pair has a characteristic Fdrster distance of about 42 A. 

The cleanest signals are generally obtained using antibodies labeled with fluorescine isothiocyanate (FITC) or carbocyanine dyes (Cy 3). If possible, avoid antibodies labeled with rhodamine (TRITC) or, to a lesser extent, Texas red, since in our hands at least, these tend to yield higher levels of background labelling For Vibratome sections, increase the incubation tune to 3-4 h. For retina or other wholemounts, incubation times on the order of 8 h may be necessary. Likewise, for cultured cells, secondary antisera incubation tunes may need to be extended, this will have to be determined empirically. 

Figure 4.4 P, profiles inside the membrane obtained for labeled BSA (BSA-FITC) after the filtration of solutions containing the labeled protein together with labeled (dextran-TRITC) and unlabeled dextran with a final concentration of 0.5 gL (a) 0.45 pm mixed esters...

Antibodies were labeled with an extrinsic fluorescent probe for the generation of a fluorescent signal during biosensor detection. The fluorescent probe used was tretramethyl-rhodamine-5-isothiocyanate (TRITC) purchased from Molecular Probes, Inc., Eugene, Oregon and stored in a desiccator at less than 0 °C prior to use. 

TRITC has been used in numerous applications involving fluorescence detection, including double-staining techniques with fluorescein-labeled probes (Mossberg and Ericsson, 1990), the synthesis of fluorescently labeled DNA probes (Smith et al., 1985), as a label in homogeneous... 

TRITC has been used in numerous applications involving fluorescence detection, including double-staining techniques with fluorescein-labeled probes (Mossberg and... 

The second label also may be a fluorescent compound, but does not necessarily have to be. As long as the second label can interact with the emission of the first label and modulate its signal, binding events can be observed. Thus, the two labeled DNA probes interact with each other to produce luminescence modulation only after both have bound target DNA and are in enough proximity to initiate energy transfer. Common labels utilized in such assay techniques include the chemiluminescent probe N-(4-aminobutyl)-N-ethylisoluminol and the fluorescent compounds FITC (Chapter 8,Section 1.1), TRITC, and Texas Red sulfonyl chloride (Chapter 8, Section 1.2). Fora review of these techniques, see Morrison (1992). 

Gottschlich et al. [26] described the separation of tetramethyl rhodamine isothiocyanate (TRITC)-labeled tryptic peptides of (3-casein. The field strength was 220 V/cm in the NCEC channel with lOmM sodium borate with 30% (v/v) acetonitrile as mobile phase. Throckmorton et al. [27] described the separation of papain inhibitor, proctolin, opioid peptide (a-casein fragment 90-95), Ile-angiotensin III and angiotensin III on a porous polymer monolith... 

FIGURE 6.10 Van Deemter plots for capillary electrophoresis ( ) and microchip electrophoresis (o), where plate height (H) of TRITC-labeled bovine serum albumin is plotted vs. migration velocity (v) [174]. Reprinted with permission from the American Chemical Society. 

FIGURE 6.40 2D separation of TRITC-labeled tryptic peptides of [S-casein. The projections of the 2D separation into the first dimension (OCEC) and second dimension (CE) are shown to the left and below the 2D contour plot, respectively. The field strengths were 220 V/cm in the OCEC channel and 1890 V/cm in the CE channel. The buffer was 10 mM sodium borate with 30% (v/v) acetonitrile. The detection point was given as y in Figure 4.16 (Chapter 4) and was 0.8 cm past valve V2 in the CE channel [333]. Reprinted with permission from the American Chemical Society. 

The red-emitting rhodamine derivatives are constructed around the same basic xanthene framework as is fluorescein (2). Tetramethyl-rhodamine isothiocyanate (TRITC) has been widely employed for immunofluorescence. Additional derivatives of rhodamine available for conjugation to antibodies include lissamine rhodamine sulfonyl chloride (RB-200-SC), rhodamine B isothiocyanate (RBITC), rhodamine X isothiocyanate (XRITC), and Texas Red (Molecular Probes, Inc.). The spectra of XRITC and Texas Red are shifted to longer wavelengths compared to those of other rhodamines, which makes them particularly useful for combination with fluorescein in dual-labeling procedures see Section 5, below). Of the two, Texas Red, which is more hydrophilic and less likely to precipitate proteins upon conjugation (12), is more commonly employed. 

Fig. 3. TRITC image of frozen tissue section treated with intratumoral injection of Rh-labeled/TATp/pFI-insensitive liposome 3(a) or Rh-labeled/TATp/pit-sensitive liposome 3(b) into LLC tumor bearing mice...

Fluorescence Microscopy. This microscopic approach is used to study the trafficking of either the vector or the nucleic acid. By a fluorescent labeling of the structure of interest (with fluorochrome such as FITC or TRITC), it is possible to visualize the internalization and trafficking processes using fluorescence microscopy or confocal microscopy. Using this approach, Midoux s group studied the internalization of polyplexes in HepG2 cells [11]. An interest of this last technique is that both the vector and the nucleic acid can be labeled. The complex evolution (and its disruption) can be observed. 

Anisotropic PS/silica hybrids containing two different fluorescent labels were reported [108] as suitable particles for biomedical applications. While the carboxy-functionalized PS part served as anchor for green fluorescing NHS-FITC, the amino-functionalized silica part was functionalized with red fluorescing TRITC. The asymmetric distribution was confirmed by confocal laser scanning microscopy. 

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