FITC is the form of fluorescein used for conjugation to all of our antibodies and purified proteins, with the exception of streptavidin. Fluorescein conjugates absorb light maximally at 492 nm and fluoresce maximally at 520 nm (Table 2 and Figure 13). FITC is a widely used fluorophore due to its long history. The major disadvantage of fluorescein is its rapid photobleaching (fading), which can be partially mitigated by the use of an anti-fading agent in the mounting medium.

DTAF (Dichlorotriazinylamino fluorescein) is another form of fluorescein, with excitation and emission peaks identical to those of FITC. JIR uses DTAF (instead of FITC) only for conjugation with streptavidin, since fluorescence from FITC is greatly quenched after conjugation with streptavidin. This phenomenon is unique to streptavidin, and is not observed with antibodies. For more information click here.

Table 1. Approximate peak wavelengths of excitation and emission for JIR conventional and DyLight fluorophores conjugated to affinity-purified antibodies. Only approximate values are given for purposes of comparing one fluorophore with another. Actual values may vary depending on the spectrofluorometer used in each laboratory.