We have observed that fluorescence from many of our fluorophore-conjugated streptavidins in solution was enhanced following the addition of a saturating amount of free biotin. Of particular significance was the difference we observed between DTAF- and FITC-conjugated streptavidins.

Fluorescence from FITC-streptavidin was extremely low when no biotin (curve F-S) was bound to the conjugate. However, after addition of a saturating concentration of free biotin (curve F-S+B) there was a 16-fold increase in fluorescence (Figure 1). A similar response from DTAF-streptavidin was less dramatic (a 1.9-fold increase)(curves D-S and D-S+B), however its fluorescence was much higher than that from FITC-streptavidin before the addition of biotin.

Figure 1

Of more importance, however, was the observation that DTAF-streptavidin was more than twice as fluorescent as FITC-streptavidin before (curves F-S and D-S) and after (curves F-S+B and D-S+B) the addition of free biotin (Figure 1). Although this difference may be somewhat less when the conjugates are bound to only one or two biotins on an antibody or other molecule, these observations suggest that DTAF-streptavidin should be a better fluorescein labeling reagent than FITC-streptavidin. We have therefore added DTAF (code number 016-010-084) to our list of streptavidin conjugates.

Unlike streptavidin fluorescein conjugates, DTAF-conjugated antibodies are the same or less fluorescent than FITC-conjugated antibodies.