Amine- and sulfhydryl-reactive derivatives of fluorescein dye, including antibody labeling kits.

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.

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.

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.

Properties of Amine-reactive Fluorescein Dyes:

Properties of Sulfhydryl-reactive Fluorescein Dyes:

Fluorescein Applications:

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

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.

Fluorescein and FITC 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 488 is a newer derivative of fluorescein, tailored for various chemical and biological applications where greater photostability and fluorescence intensity, pH independence, and a narrower emission spectrum are required.

DyLight 488 Labeling Kits
DyLight 488 and eight other DyLight Reactive Fluors
Rhodamine Dye and Labeling Kits
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 46409 NHS-Fluorescein 1 g $412.00 Add 46410 NHS-Fluorescein Replaces Product No. 46100 100 mg $64.00 Add 46424 FITC Replaces Product No. 46110 1 g $155.00 Add 46425 FITC 100 mg $52.00 Add 53027 FITC 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. 53004 Kit contains: FITC, 3 vials Borate Buffer, 1 ml Purification Resin, 5 ml Spin Columns, 6 each Collection Tubes, 12 each Kit $247.00 Add 53029 NHS-Fluorescein 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. 53000 Kit contains: NHS-Fluorescein, 3 vials Borate Buffer, 1 ml Purification Resin, 5 ml Spin Columns, 6 each Collection Tubes, 12 each Kit $247.00 Add 46130 Fluorescein-5-maleimide 25 mg $240.00 Add 46120 5-Iodoacetamido-fluorescein (5-IAF) 25 mg $275.00