TRITC rhodamine

NHS-rhodamine is an amine-reactive fluorescent probe that contains a carboxy-succinimidyl ester group off the No. 5 or 6 carbons on rhodamine s lower-ring structure (Kellogg et al., 1988). The 5- and 6-isomers are virtually identical in their reactivity and fluorescent characteristics. Similar to TRITC (described previously), NHS-rhodamine can be used to label proteins and other macromolecules that contain primary amine groups. The isomeric forms of the fluorescent probe are available in mixed and purified forms (Invitrogen, Thermo Fisher). The pure forms are... 

The fluorescent properties of NHS-rhodamine are similar to TRITC. The wavelength of maximal absorbance or excitation for the reagent is 544 nm and its emission maximum is 576 nm, exhibiting a visual color of orange-red. Its molar extinction coefficient at 546 nm in a methanol environment is 63,000M 1cm 1. Other components in solution as well as the pH (in aqueous buffers) can change this value. 

The spectral characteristics of protein conjugates made with Lissamine rhodamine B derivatives are of longer wavelength than those of tetramethylrhodamine—more toward the red region of the spectrum. In addition, modified proteins have better chemical stability and are somewhat easier to purify than those made from TRITC (discussed previously). Lissamine derivatives also make more photostable probes than the fluorescein derivatives (Section 1, this chapter). 

ABEI, M(4-ami nobutyl )-Methylisolu mi nol BSA, bovine serum albumin CL, chemiluminescence DNPO, tas-(2,4-dinitrophenyl)oxalate ECL, electrogenerated chemiluminescence EMMA, electrophoretically mediated microanalysis EY, eosine Y FR, lluorescamine HRP, horseradish peroxidase ILITC, isoluminol isothiocyanate LOD, limit of detection RITC, rhodamine B isothiocyanate TCPO, Mv-(2,4,6-trichlorophenyl)oxalate TEA, triethylamine TRITC, tetramethylrhodamine isothiocyanate. 

Consequently, the research work of Hara s group continued focusing on the improvement of protein determination using CE combined with online CL detection. By replacing EY by the Rhodamine B isothiocyanate (RITC) dye in the binary complexes formed with the proteins BSA or human serum albumin (HSA) and using a different imidazole buffer solution of pH 6, the sensitivity was increased [72], However, best detection limits for these determinations were found employing the tetramethylrhodamine isothiocyanate isomer (TRITC) dye, left for 4 h with a standard solution of BSA in acetonitrile followed by introduction into the capillary. For BSA, a detection limit of 6 nM was reached [73],... 

Figure 14.2. Chemical structures of some commonly used organic fluorescent probes 1, fluorescein-5-isothiocyanate (FITC) 2, tetramethylrhodamine-5-isothiocyanate (TRITC) 3, 5-carboxyrhodamine B 4, rhodamine X isothiocyanate (XRITC) 5, malachite green isothiocyanate 6, eosin-5-isothiocyanate 7, 1-pyreneisothiocyanate 8, 7-dimethylaminocoumarin-4-acetic acid 9, CY5.180Su.

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. 

Abbreviations AMCA, 7-amino-4-methylcoumarin B-PE, B phycoerythrin Cy, cyanine DAMC, diethylaminocoumarin FITC, fluorescein isothiocyanate RB-200-SC, lissamine rhodamine sulfonylchloride R-PE, R phycoerythrin SITS, 4-acetamido-4 -isothiocyanato-stilbene-2,2 -disulfonic acid TRITC, tetramethyl rhodamine isothiocyanate XRITC, rhodamine X isothiocyanate. Information obtained from refs. 2, 9, and 10. 

The red-emitting rhodamine derivatives are constructed around the same basic xanthene framework as is fluorescein (2). Tetramethylrhodamine 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... 

Recently, cyanine fluorochromes covering a wide spectral range have become available for immunofluorescence (13,14). The red-emitting fluoro-chrome Cyanine 3.18, which was shown to give a significantly brighter image than TRITC, lissamine rhodamine, Texas Red, or fluorescein under specific conditions of microscopy (7), provides a useful alternative to the rhodamines. Other useful substitutes for the rhodamines include the BODIPY TR and TMR, and Alexa 568 and 594 fluorochromes. The latter are newly introduced and appear to offer superior photostability. 

The second strategy uses combinations of different antibodies coupled to fluorochromes with distinct emission maxima (5,9). The most relevant fluoro-chromes for combined antigen detection are fluorescein isothiocyanate (FITC abs. max. 494 nm, emiss. max. 517 nm), rhodamine isothiocyanate (TRITC ... 

The fluorescent properties of TRITC (mixed isomers) include an absorbance maximum at about 544 nm and an emission wavelength of 570 nm. Fluorescent quenching of the molecule is possible. Under concentrated conditions, rhodamine-to-rhodamine interactions result in self-quenching, which reduces its luminescence yield. This phenomenon can occur with TRITC-tagged molecules, as well. If derivatization of a protein is done at too high a level, the resultant quantum yield of the conjugate will be depressed from expected values. Typically, modifications of proteins involve adding no more than 8-10 rhodamine molecules per molecule of protein, with a 4-5 substitution level considered optimal. 

This approach has been shown to work with a number of different fluorescent probes such as the short-wavelength fluorophores dansyl sul-fonyl chloride and coumarin chloride and the long-wavelength fluorophores tetramethylrhodamine-5-(and-6)-isothiocyanate [5(6)-TRITC], 5-(and-6)-carboxytetramethylrhodamine, succinimidyl ester [5(6)-TAMRA, succin-imidyl ester] and lissamine rhodamine B sulfonyl chloride (each in conjunction with different binding functionalities on the SAM surface. 

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... 

Fluorescein isothiocyanate (FITC) or tetramethyl-rhodamine isothiocyanate (TRITC). 

Abbreviations used FITC = fluorescein isothiocyanate DTAF = dichloro-triazinylaminofluorescein, TRITC = tetramethylrhodamine isothiocyanate, RB-200SC s lissamine rhodamine sulfonylchloride, RBITC = rhodamine B isothiocyanate, XRITC = rhodamine X isothiocyanate CY3.18 = cyanine 3.18, B-PE = B phycoerythrin, R-PE = R phycoerythrin SITS = 4-acetamido-4 -isothiocyanatostilbene-2,2 -disulfonic acid, DAMC = diethylaminocoumarin, AMCA = 7-Amino-4-methylcoumarin-3-acetic acid. Information obtained from refs. (2,9,10)... 

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. 

Fluorescence resonance energy transfer (FRET) is a technique that has been used to measure distances between pairs of proximal fluorochromes. A suitable pair consists of a donor fluorochrome, which has an emission spectrum that significantly overlaps with the absorption spectrum of an acceptor fluorochrome (2). With the availability of monoclonal antibodies to many cell-surface determinants, intramolecular distances between nearby epitopes and intermolecular distances between adjacent cell-surface macromolecules can be investigated to analyze molecular interactions influencing important cellular events. Such monoclonal antibodies can be conjugated to fluorescein-isothiocyanate (FITC) as the donor, and either tetramethyl-rhodamine-isothiocyanate (TRITC) or phycoerythrin (PE) as the acceptor. 

Individual coverslips are mounted cell-side down onto fresh glass slides with PBS see Note 7). Cells are viewed with a Nikon Eclipse E400 microscope under bright light or under epifluorescence with rhodamine/TRITC filter (58) (see Note 8). 

Mounted slides were observed with the Nikon Eclipse microscope under the bright light or under the epi-fluorescence using Rhodamine/TRITC filter (Fig. 1). 

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. 

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