TRITC and Rhodamine Dyes and Labeling Kits

Thermo Scientific Rhodamine Labeling Reagents and Kits are high-performance derivatives of rhodamine dye, activated for easy and reliable labeling of antibodies, proteins and other molecules for use as fluorescent probes.

Tetramethylrhodamine isothiocyanate (TRITC) and NHS-Rhodamine are amine-reactive derivatives of rhodamine dye that have wide-ranging application as antibody and other probe labels for use in fluorescence microscopy, flow cytometry and immunofluorescence-based assays such as Western blotting and ELISA. Choose stand-alone packages of either TRITC or NHS-Rhodamine, or select a convenient Antibody Labeling Kit with either fluorescent reagent.

Highlights:

• Easy – convenient kit with NHS-rhodamine to label and purify antibody in about one hour
• Amine-specific labeling – NHS-ester and TRITC varieties of rhodamine efficiently label antibodies and other purified proteins at primary amines (lysine side chains)
• Optimized procedure – following the standard protocol results in antibodies with excellent dye:protein ratios for optimum activity and fluorescence
• Single-use fluors – no need to weigh tiny amounts powder; kit contains single-use vials of reagent
• Efficient purification – kit includes purification resin and easy-to-use spin columns, ensuring rapid and efficient removal of non-reacted dye and excellent protein recovery

Applications:

• Label antibodies for use as immunofluorescent probes
• Label oligonucleotides for hybridization probes
• Detect proteins in gels and on Western blots

Properties of Rhodamine Dyes:

Chemical structures of TRITC and NHS-Rhodamine. Both of these compounds allow fluorescent labeling of primary amines on proteins and other molecules. See our review of Amine-Reactive Crosslinker Chemistry.

Thermo Scientific Pierce Rhodamine Dyes are mixtures of isomers with reactive groups attached at the 5- and 6-positions of the bottom ring. The properties of these isomers are indistinguishable in terms of excitation and emission spectra, and for protein applications there is no need to isolate a specific isomer.

TRITC is the base tetramethylrhodamine molecule functionalized with an isothiocyanate reactive group (–N=C=S) at one of two hydrogen atoms on the bottom ring of the structure. This derivative is reactive towards primary amine groups on proteins, peptides and other biomolecules. NHS-Rhodamine is activated with the N-hydroxy-succinimidyl-ester (NHS ester) functional group. Compared to TRITC, the NHS-ester deriviative has greater specificity toward primary amines in the presence of other nucleophiles and results in a more stable linkage following labeling. Texas Red* Sulfonyl Chloride is a long-wavelength derivative of rhodamine that is modified with sulfonyl chloride for reaction to primary amines. We recommend DyLight 594 NHS Ester as an alternative to Texas Red Sulfonyl Chloride because it is brighter, more photostable and has a more specific labeling chemistry that can be used at physiologic pH.

Application Data:

A. Rhodamine	B. Hoechst	C. Merged
Detection of a-tubulin in A549 cells demonstrates use of rhodamine-labeled secondary antibody. Cells were grown in 96-well microplates for 18-20 hrs, fixed with 4% paraformaldehyde (Product #28906) and permeabilized with 0.1% Surfact-Amps X-100 (Product #28314). Cells were then probed with a mouse anti-a-tubulin primary antibody (0.4µg/ml) and Rhodamine-goat anti-mouse secondary antibody (2µg/ml). Nuclei were labeled with Hoechst Dye. Images were acquired by fluorescence microscopy. A. Fluorescence image shows a delicate network of a-tubulin (pseudo-colored green) located exclusively in the cytoplasm. B. Nuclear counterstain with Hoechst Dye (pseudo-colored blue) C. Merged image.

General References:

1. Miki, M. and dos Remedios, C.G. (1988). Fluorescence quenching studies of fluorescein attached to lys-61 or cys-374 in actin: effects of polymerization, myosin sub fragment-1 binding, and tropomyosin-troponin binding. J. Biochem. 104, 232-235.
2. Smith, L.M., et al. (1987). The synthesis and use of fluorescent oligonucleotides in DNA sequence analysis. Meth. Enzymol. 155, 260-301.
3. Vera, J.C., et al. (1988). Purification, amino terminal analysis and peptide mapping of proteins after in situ postelectrophoretic fluorescent labeling. Anal. Biochem. 174, 38-45.
4. Szewczyk, B. and Summers, D.F. (1987). Fluorescent staining of proteins transferred to nitrocellulose allowing for subsequent probing with antisera. Anal. Biochem. 164, 303-306.
5. Der-Balian, G.P., et al. (1988). Fluorescein labeling of Fab while preserving single thiol. Anal. Biochem. 173, 59-63.
6. Vigers, G.P.A., et al. (1988). Fluorescent microtubules break up under illumination. J. Cell Biol. 107, 1011.
7. Goding, J.W. (1976). Conjugation of antibodies with fluorochrome: modifications to the standard methods. J. Immunol. Meth. 13, 215-226.
8. Szewczyk, B., et al. (1987). Use of different fluorochromes for monitoring protein elution and transfer. Electrophoresis 8, 25-28.
9. Smith, L.M., et al. (1986). Fluorescence detection in automated DNA sequence analysis. Nature 321, 674-678.
10. Staines, W.A., et al. (1988). Three-color immunofluorescence histochemistry allowing triple labeling within a single section. J. Histochem. Cytochem. 36(2), 145-151.

Related Resources:

Review of Fluorescent Probes
Tech Tip #31 – Calculate dye:protein (F/P) molar ratios

Related Products:

DyLight Fluor Labeling Reagents and Kits
Fluorescent Labeling – Top-level menu of all reagents