Amine-reactive derivatives of rhodamine dye and associated fluorescent labeling kits.

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.

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.

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.

Properties of Rhodamine Dyes:

Rhodamine Applications:

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

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.

Rhodamine and TRITC Labeling Kit Highlights:

• Fast – label and purify antibody in about one hour
• Amine-specific labeling – the NHS-ester or isothiocyanate efficiently labels 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; kits contain single-use vials of reagent
• Efficient purification – kits include purification resin and easy-to-use spin columns, ensuring rapid and efficient removal of non-reacted dye and excellent protein recovery

Related Products and Resources

DyLight 549 and DyLight 594 Fluors are newer derivatives of rhodamine and Texas Red Dyes, tailored for various chemical and biological applications where greater photostability and fluorescence intensity, pH independence, and a narrower emission spectrum are required.

Fluorescein Dye and Labeling Kits
DyLight 549 and DyLight 594 Labeling Kits
DyLight 549 and eight other DyLight Reactive Fluors
Calculate dye:protein (F/P) molar ratios (Tech Tip #31)
Top menu of all Fluorescent Labeling Reagents, including AMCA, DAPI and R-Phycoerythrin

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.

Thermo Scientific Pierce Fluorescent Dyes are available in bulk quantities and prices.

Ordering Information

Certificate of Analysis   Product Instructions   MSDS

Buy Product # Description Pkg. Size Files Price Add 46112 TRITC 10 mg $225.00 Add 46406 NHS-Rhodamine Replaces Product No. 46102 25 mg $102.00 Add 53031 NHS-Rhodamine Antibody Labeling Kit Sufficient reagents to label and purify 3 x 1 mg (2 mg/ml) of IgG or similar amounts of other proteins. Replaces Product No. 53002 Kit contains: NHS-Rhodamine, 3 vials Borate Buffer, 1 ml Purification Resin, 5 ml Spin Columns, 6 each Collection Tubes, 12 each Kit $247.00 Add 46115 Texas Red Sulfonyl Chloride DyLight 594 NHS Ester is a superior alternative to Texas Red Sulfonyl Chloride 10 x 1 mg $228.00