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TRITC and Rhodamine Dyes and Labeling Kits


Amine-reactive rhodamine dyes and complete fluorescent labeling kits.

Rhodamine Labeling Reagents and 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 structure of NHS-Rhodamine Chemical structure of tetramethyl rhodamine isothiocyanate, TRITC
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.

  NHS-Rhodamine TRITC Texas Red* Sulfonyl Chloride
Alternative names 5/6-carboxy-tetramethyl-rhodamine succinimidyl ester, 5/6-TAMRA SE 5(6)-tetramethyl-rhodamine isothiocyanate mixed isomer, 5/6-TRITC, TMR Sulforhodamine 101 sulfonyl chloride, Texas Red-X, T1905, T353
Molecular weight 527.5 479.0 (chloride salt);
443.5 (no Cl)
625.15
Excitation source 546nm spectral line, mercury-arc lamp; 543nm spectral line, He-Ne laser 546nm spectral line, mercury-arc lamp; 543nm spectral line, He-Ne laser 568nm spectral line, Ar-Kr laser; 594nm spectral line orange He-Ne laser
Excitation wavelength 552nm 544nm 596nm
Emission wavelength 575nm 572nm 615nm
Extinction coefficient >80,000/M cm >80,000/M cm ~80,000/M cm
CAS # 246256-50-8 95197-95-8 82354-19-6
Purity > 90% by HPLC > 85% by HPLC > 85% by HPLC
Solubility Soluble in DMF or DMSO Soluble in DMF or DMSO Soluble in DMF or methylene chloride (not stable in DMSO)
Storage Desiccated at -20°C,
protect from moisture,
use only fresh solutions
Desiccated at -20°C,
protect from moisture,
use only fresh solutions
Desiccated at -20°C,
protect from moisture,
use only fresh solutions
Reactive groups NHS ester, reacts with primary amines at pH 7.0 to 9.0 Isothiocyanate, reacts with primary amines at pH 7.0 to 9.0 Sulfonyl chloride, reacts with primary amines at pH 7.0 to 9.0

Application Data:


Detection of alpha-tubulin in A549 cells using secondary antibody labeled with Thermo Scientific Pierce NHS-Rhodamine.
A. Rhodamine

 

A549 cells stained with Hoechst dye.
B. Hoechst

 

Merged image of nucleus and alpha-tubulin staining in A549 cells using secondary antibody labeled with Thermo Scientific Pierce NHS-Rhodamine.
C. Merged

 

Detection of α-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 (Part No. 28906) and permeabilized with 0.1% Surfact-Amps X-100 (Part No. 28314). Cells were then probed with a mouse anti-α-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 α-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:

DyLight 550 and DyLight 594 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.

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


Ordering Information
 
Product # Description Pkg. Size Instructions MSDS CofA Price
46112 TRITC
Formulation: Tetramethylrhodamine isothiocyanate fluorescent dye
Sufficient For: Approx. 5 uses to label a total of 139mg of IgG using typical conditions
10mg Product Instructions for product #46112 TRITC MSDS for product #46112 TRITC Certificate of Analysis for product #46112 TRITC $250.00

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46406 NHS-Rhodamine
Formulation: N-hydroxysuccinimide ester activated rhodamine fluorescent dye
Sufficient For: Approx. 12 uses to label a total of 316mg of IgG using typical conditions
25mg Product Instructions for product #46406 NHS-Rhodamine MSDS for product #46406 NHS-Rhodamine Certificate of Analysis for product #46406 NHS-Rhodamine $115.00

Add to cart
53031 NHS-Rhodamine Antibody Labeling Kit
Formulation: Multi-component kit with rhodamine fluorescent dye
Sufficient For: Labeling and purifying 3 x 1mg (2mg/mL) of IgG or other proteins

Kit Contents:
NHS Rhodamine, 3 x 35μg vials
Borate Buffer, 1mL
Purification Resin, 5mL
Spin Columns, 6 each
Collection Tubes, 12 each
3-rxn kit Product Instructions for product #53031 NHS-Rhodamine Antibody Labeling Kit MSDS for product #53031 NHS-Rhodamine Antibody Labeling Kit Certificate of Analysis for product #53031 NHS-Rhodamine Antibody Labeling Kit $282.00

Add to cart
46115 Texas Red Sulfonyl Chloride
Formulation: Sulfonyl chloride activated derivative of rhodamine fluorescent dye
Sufficient For: Approx. 5 uses to label a total of 10mg of IgG using typical conditions

Note: DyLight 594 NHS Ester is a superior alternative to Texas Red Sulfonyl Chloride.
10mg Product Instructions for product #46115 Texas Red Sulfonyl Chloride MSDS for product #46115 Texas Red Sulfonyl Chloride Certificate of Analysis for product #46115 Texas Red Sulfonyl Chloride $255.00

Add to cart

Instructions | MSDS | CofA
Product Instructions | MSDS | CofA  

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