Large Stokes shift (the difference in wavelength between positions of the band maxima of the absorption and emission spectra)
* Data as DTBTA-Eu3+
‡The fluorescence intensity of DTBTA-Eu3+ is very weak (fluorescence quantum yield: 9.1%), and if the solution concentration is low, the fluorescence itself cannot be observed by eye. Be sure to use an analytical instrument capable of time-resolved fluorescence measurement.
Historically a wide variety of organic fluorescent reagents have been used as tags or labels, such as fluorescein, rhodamine, and various cyanin dyes. Different from these organic reagents, certain lanthanide complexes, especially those of Eu3+ and Tb3+, are also recognized as efficient fluorescent labels, owing to their distinct properties specific to lanthanide complexes; they are excited in the UV region (310-340nm) and emit fluorescence at ca. 615nm (Eu3+) and ca. 545nm (Tb3+), with the long lifetimes of several hundred microseconds to more than 1 milliseconds. By taking advantage of these properties, time-resolved fluorometric measurement can remove background fluorescence from the sample matrix and often gives detectability better than one order of magnitude compared to those of conventional fluorometric assays. The other reagent, ATBTA-Eu3+, has an amino group instead of dichlorotriazinyl in DTBTA-Eu3+, and is more stably stored, since it does not have the hydrolysable dichlorotriazinyl group. DTBTA-Eu3+ can be directly labeled to amino groups of biomolecules, whereas ATBTA-Eu3+ is used as a label after conversion to DTBTA-Eu3+ by reacting with trichlorotriazene. Scheme 1 summarizes these reactions and the labeling of DTBTA-Eu3+ to the primary amine groups of proteins and nucleic acids. Although ATBTA-Eu3+ is not so strongly fluorescent as DTBTA-Eu3+, the fluorescence becomes strong after reaction with trichlorotriazene. The fluorescence spectrum of ATBTA-Eu3+ is basically the same with that of DTBTA-Eu3+.
The new lanthanide chelate reagent, DTBTA-Eu3+, has a high stability constant, and therefore the problem of fluorescence intensity change in different buffers has been greatly reduced. DTBTA-Eu3+ has also several advantages such as the intensity stability in water for a long period, and the stability against photo-bleaching. The excitation and emission spectra are shown in Fig. 1.
Typical Procedure: Preparation of DTBTA-Eu3+ Dissolve 2mg of ATBTA-Eu3+ in 60μL of 0.1M acetate buffer (pH4.9). This solution is added 0.43mg of Cyanuric Chloride in 25μL of acetone, and stirred for 30 min. The reaction mixture is added dropwise to 1mL of acetone, and formed precipitate is centrifuged. After washing with 0.5mL of acetone twice, the yellow powder is dried in vacuum for 1 h. Dissolve the powder in 1mL of carbonate buffer gives (pH9) for labeling. This solution contains ca. 2mM of labeling reagent.
Warning This labeling reagent is deactivated by hydrolysis, especially in alkali solution. The reagent dissolved in water should be used immediately. For temporary storage, the reagent should be dissolved in buffer solution at acidic pH (pH~5) and kept at 0ºC.
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