TechFAQ #6

Why is DTAF-conjugated Streptavidin offered
instead of FITC-conjugated Streptavidin?

Technical Service e-mail
tech@jireurope.com tech@jacksonimmuno.com

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.

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).

Figure 1. Comparison of fluorescence between FITC-streptavidin (F-S) and DTAF-streptavidin (D-S) before and after (F-S+B and D-S+B) the addition of free biotin. Spectra were obtained with an M-series spectro-fluorometer system from Photon Technologies International, Inc.

Figure 2. Human peripheral lymphocytes were stained with anti-CD3, biotin-SP-goat anti-mouse IgG (H+L) (min X Hu, Bov, Hrs Sr Prot) (115-065-062), and either FITC-streptavidin (blue) or DTAF-streptavidin (green). Stained cells were analyzed in a FACSCalibur (Becton Dickinson) flow cytometer.

Although this difference may be somewhat less apparent when the conjugates are bound to only one or two biotins on an antibody, these observations suggest that DTAF-streptavidin should be a better fluorescein labeling reagent than FITC-streptavidin. In fact as Figure 2 shows, when the two conjugates were compared for brightness in a flow cytometry experiment, DTAF-streptavidin was about two and a half times brighter than FITC-streptavidin. For this reason, JIR offers only DTAF- (rather than FITC-) conjugated streptavidin. This phenomenon is unique to streptavidin, and is not observed with antibodies.