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Technical Information on Probes Conjugated to Affinity-Purified Antibodies and to Other Proteins: Fluorescein Isothiocyanate (FITC) |
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Fluorescein conjugates absorb light maximally at 492 nm and fluoresce maximally at 520 nm (Table 1 and Figure 2). FITC is a widely used fluorophore because it has been available for a long time and it has a high quantum yield. The major disadvantage of fluorescein is rapid photobleaching (fading), which can be mitigated in the presence of an anti-fading reagent. FITC is the form of fluorescein used for the majority of our conjugates. Dichlorotriazinylamino fluorescein (DTAF) is another derivative of fluorescein, with excitation and emission peaks identical to those of FITC. DTAF is preferred over FITC for conjugates with streptavidin only, because of a significant difference in brightness on streptavidin. Table 1: Approximate* peak wavelengths of absorption and emission for different fluorophore-conjugated, affinity-purified antibodies.
*Only approximate values are given for purposes of comparing one fluorophore with another. |
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Figure 2. Excitation and emission spectra of different fluorophore-conjugated, affinity-purified antibodies. This figure illustrates the relative shape and position of each fluorophore in the peak region of its excitation and emission following conjugation to antibodies. Quantitative comparisons should not be made since peak heights have been normalized. All spectra were obtained with a M-Series spectrofluorometer system from Photon Technology International, Inc. |
