FITC and Fluorescein Dyes and Labeling Kits

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

Fluorescein isothiocyanate (FITC) and NHS-Fluorescein are amine-reactive derivatives of fluorescein 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. Fluorescein-5-maleimide and 5-IAF are sulfhydryl-reactive derivatives of fluorescein dye. Choose from stand-alone packages of these labeling reagents or select a convenient FITC or NHS-Fluorescein Antibody Labeling Kit.

Highlights:

• Easy – convenient kits with FITC or NHS-fluorescein to label and purify antibody in about one hour
• Amine-specific labeling – NHS-ester and isothiocyanate varieties of fluorescein efficiently label antibodies and other purified proteins at primary amines (lysine side chains)
• Optimized kit 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 of 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
• Sulfhydryl-specific reagents, too – maleimide and iodoacetyl varieties label proteins and other molecules having free thiols (cysteine side chains)

Applications:

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

Amine-reactive Fluorescein Dyes:

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

FITC is the base fluorescein 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-fluorescein is activated with the N-hydroxy-succinimidyl-ester (NHS ester) functional group. Compared to FITC, 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. Pierce Amine-reactive Fluorescein 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.

Sulfhydryl-reactive Fluorescein Dyes:

Chemical structures of Fluorescein-5-maleimide and 5-IAF. Both of these compounds allow fluorescent labeling of sulfhydryl groups on proteins and other molecules. See our review of Sulfhydryl-Reactive Crosslinker Chemistry.

Fluorescein-5-maleimide and 5-IAF are sulfhydryl-reactive derivatives of fluorescein dye. Fluorescein-5-maleimide is the base fluorescein molecule functionalized with a maleimide reactive group by replacing a hydrogen atom on the bottom ring of the structure. 5-IAF is the core fluorescein molecule functionalized with an iodoacetamide group. Both fluorescein derivatives are reactive toward sulfhydryl groups (e.g., reduced cysteine residues) on proteins, peptides and other biomolecules.

Application Data:

A. Fluorescein	B. Hoechst	C. Merged
Detection of a-tubulin in A549 cells demonstrates use of fluorescein-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 Fluorescein-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:

Anti-FITC Monoclonal Antibody (Part No. MA5-14696)
DyLight 488 Antibody Labeling Kits
DyLight 488 and other DyLight Reactive Fluors
Fluorescent Labeling – Top-level menu of all reagents