5-FITC

CAS#：3326-32-7

5-FITC；5-Flourescein iso-thiocyanate；FITC 'Isomer I' [5-FITC; fluorescein-5-isothiocyanate]

Fluorescent Dye Isothiocyanates 

Isothiocyanates form thioureas upon reaction with amines. It is proven that some thiourea products (in particular, the conjugates from a-amino acids/peptides/proteins) are much less stable than the conjugates that are prepared from the corresponding succinimidyl esters. It has been reported that antibody conjugates prepared from fluorescein isothiocyanates deteriorate over time. We strongly recommend that you use succinimidy esters for your conjugations whenever possible. There are few factors that need be considered when SE compounds are used for conjugation reaction: 

1). Solvents: For the most part, reactive dyes are hydrophobic molecules and should be dissolved in anhydrous dimethylformamide (DMF) or dimethylsulfoxide (DMSO). 

2). Reaction pH: The labeling reactions of amines with isothiocyanates are strongly pH dependent. Isothiocynate reagents react with non-protonated aliphatic amine groups, including the terminal amines of proteins and the e-amino groups of lysines. Protein modifications by isothiocyanates may require a pH 9.0-10.0 for optimal conjugations.

 3). Reaction Buffers:Buffers that contain free amines such as Tris and glycine must be avoided when using an amine-reactive reagent. Ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for protein precipitation must also be removed before performing dye conjugations. High concentrations of nucleophilic thiol compounds should also be avoided because they may react with the labeling reagent to form unstable intermediates that could destroy the reactive dye. 

4). Reaction Temperature: Isothiocynate conjugations are usually done at room temperature. However, either elevated or reduced temperature may be required for a particular labeling reaction.

FITC 'Isomer I' [5-FITC; fluorescein-5-isothiocyanate]

Features and Biological Applications

Despite the availability of alternative amine-reactive fluorescein derivatives that yield conjugates with superior stability and comparable spectra, fluorescein isothiocyanate (FITC) remains one of the most popular fluorescent labeling reagents, probably due to the low material cost.  It appears that 5-FITC is more widely used than the 6-FITC isomer. FITC reagents are prominently used to label proteins. In addition, FITC has also been used to label peptides, oligonucleotides and other small organic ligands.  Cautions must be exercised for the storage of FITC conjugates.

References

1.  Hattori S, et al. (2002). Real-time zymography and reverse zymography: a method for detecting activities of matrix metalloproteinases and their inhibitors using FITC- labeled collagen and casein as substrates. Anal Biochem 301, 27-34.

2.   Thiagarajah JR, et al. (2001). Evidence of amiloride-sensitive fluid absorption in rat descending colonic crypts from fluorescence recovery of FITC-labeled dextran after photobleaching. J Physiol 536, 541-53.