Tetramethylrhodamine

Fig. 1 Examples of the structures of a few slow-response electric field sensitive dyes 3, 3 -dipropylthiadicarbocyanine (DiSC3(5)), tetramethylrhodamine methyl ester (TMRM), and bis-(3-propyl-5-oxoisooxazol-4-yl)pentamine oxonol (oxonol VI)...

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.

Fig. 6.3. Chemical structures of rhodamine and some derivatives. TAMRA = N,N,TV,A-tetramethylrhodamine. Lissamine rhodamine = 3,5-disulfonyl-N,N, A,A-tetramethylrhodamine.

A different strategy for measuring protease activity is based on the property of xanthene dyes to form H-type dimers (see Sect. 6.2.3) when they are in close proximity. These dimers are accompanied with a characteristic quenching of their fluorescence and, particularly for rhodamines, with a blue shift in the absorption spectrum [121, 122]. The probe D-NorFES-D designed to measure activity of elastase in HL-60 cells consists of an undecapeptide derivatized with one tetramethylrhodamine dye on each side. The sequence contains proline residues to create a bent structure and bring the two fluoro-phores in close proximity. Intact D-NorFES-D shows 90% of its fluorescence quenched plus a blue shift of the absorption spectrum. After addition of the serine protease elastase, an increase in the fluorescence and a bathochromic shift of the absorption spectrum is observed, resulting in an increase in the emission ratio [80],... 

Tetramethylrhodamine-5-(and 6)-isothiocyanate (TRITC) is one of the most popular fluorescent probes available. The isothiocyanate derivative of tetramethylrhodamine is synthesized by... 

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). 

The iodoacetamido derivatives of tetramethylrhodamine possess a sulfhydryl-reactive iodoacetyl group (Chapter 1, Section 4.2 and Chapter 2, Section 2.1) at either the 5- or 6-carbon position on their lower ring. The isomers are commercially available only in mixed form, but some reactivity and specificity differences between the purified 5- and 6-derivatives toward various sulfhydryl sites in proteins may be observed (Ajtai et al., 1992) (Invitrogen). 

The spectral properties of these derivatives are similar to native rhodamine. The excitation maximum occurs at about 543 nm and its emission peak at 567nm, producing light in the orange-red region of the spectrum. The extinction coefficient of tetramethylrhodamine-5-(and-6)-iodoacetamide in methanol at its wavelength of maximum absorptivity, 542 nm, is 81,000M-1cm-1. 

Figure 9.18 This iodoacetamide derivative of tetramethylrhodamine can be used to label sulfhydryl groups via thioether bond formation.

The following protocol for labeling proteins with tetramethylrhodamine-5-(and-6)-iodoa-cetamide represents a general guideline. The procedure should be optimized for each macromolecule being labeled to obtain the best F/P ratio to produce intense fluorescence and high activity in the final complex. 

Prepare a 20mM tetramethylrhodamine-5-(and-6)-iodoacetamide solution by dissolving 11.3 mg/ml of DMF. Prepare fresh and protect from light. 

Texas Red hydrazide is a derivative of Texas Red sulfonyl chloride made by reaction with hydrazine (Invitrogen). The result is a sulfonyl hydrazine group on the No. 5 carbon position of the lower-ring structure of sulforhodamine 101. The intense Texas Red fluorophore has a QY that is inherently higher than either the tetramethylrhodamine or Lissamine rhodamine B derivatives of the basic rhodamine molecule. Texas Red s luminescence is shifted maximally into the red region of the spectrum, and its emission peak only minimally overlaps with that of fluorescein. This makes derivatives of this fluorescent probe among the best choices of labels for use in double-staining techniques. 

In the preparation of 15 nm core-shell fluorescent silica particles, Ow et al. (2004) reported that the naked core (2.2 nm) alone produced a fluorescence intensity of less than the free dye in solution, presumably due to dye quenching. However, upon addition of the outer silica shell around the core, the brightness of the particles increased to 30 times that of the free dye (using tetramethylrhodamine-5-(and 6)-isothiocyanate (TRITC)). They speculate that shell may protect the core from solvent effects, as evidenced by a lack of spectral shift upon changing the solvent in which the particles are suspended. 

Ajtai, K. (1992) Stereospecific reaction of muscle fiber proteins with the 5 or 6 iodoacetamido derivative of tetramethylrhodamine Only the 6 isomer is mobile on the surface of SI. Biophys. J. 61, A278, Abstract 1647. 

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],... 

TEP TL TLC TMB TMP TMP TMPG TNS TPB TRIS TRITC TTAB UA USDA-FSIS Triethylphosphine Total luminescence Thin layer chromatography T etramethylbenzidine 2,4,6,8-Tetrathiomorpholinopyrimido [5,4-d] pyrimidine Trimethylphosphine S S -Trimethoxyphenylglyoxal Potassium 2-p-toluidinylnaphthalene-6-sulfonate Tetradecylpyridine bromide Tris (hydroxymethyl) aminomethane Tetramethylrhodamine isothiocyanate Tetradecyltrimethyl ammonium bromide Uric acid U.S. Department of Agriculture-Food Safety and Inspection Service... 

LOD is defined as the lowest concentration of an analyte that produces a signal above the background signal. LOQ is defined as the minimum amount of analyte that can be reported through quantitation. For these evaluations, a 3 x signal-to-noise ratio (S/N) value was employed for the LOD and a 10 x S/N was used to evaluate LOQ. The %RSD for the LOD had to be less than 20% and for LOQ had to be less than 10%. Table 6.2 lists the parameters for the LOD and LOQ for methyl paraben and rhodamine 110 chloride under the conditions employed. It is important to note that the LOD and LOQ values were dependent upon the physicochemical properties of the analytes (molar absorptivity, quantum yield, etc.), methods employed (wavelengths employed for detection, mobile phases, etc.), and instrumental parameters. For example, the molar absorptivity of methyl paraben at 254 nm was determined to be approximately 9000 mol/L/cm and a similar result could be expected for analytes with similar molar absorptivity values when the exact methods and instrumental parameters were used. In the case of fluorescence detection, for most applications in which the analytes of interest have been tagged with tetramethylrhodamine (TAMRA), the LOD is usually about 1 nM. 

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.

Dichlorotriazinofluorescein-goat anti-mouse IgG, bound to tetramethylrhodamine in PBS solution 2.58 (76%) 0.63 (24%) PMFS 107... 

An evanescent wave fiber optic immunosensor has been used for the detection of ricin in river water.(131) A tapered fiber optic waveguide with covalently bound anti-ricin IgG is used in a sandwich format, with tetramethylrhodamine-labeled antibody as tracer. In a two-step format, ricin in the sample is bound to the fiber first, and then the fiber is exposed to the tracer antibody. Sensitivity is 1 ng/ml for the two-step assay. The one-step assay, in which the fiber optic probe contacts the sample and labeled antibody simultaneously is less sensitive, but more convenient. 

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... 

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