TechFAQ #6
Why is DTAF-conjugated Streptavidin offered
instead of FITC-conjugated Streptavidin?
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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).
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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. |
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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. |
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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. |
