Fluorophores conjugated to avidin and streptavidin may be quenched significantly, apparently because the dyes interact with amino acid residues in the biotin-binding pocket. Exceptions include Cascade Blue– and phycobiliprotein-labeled avidin and streptavidin; the dyes in these conjugates are not quenched because they do not interact with the biotin-binding site. A significant recovery of the avidin or streptavidin conjugate's fluorescence can be obtained if biotin is added as a final incubation step in the staining procedure (see figure). Fluorescence enhancement of avidin conjugates by biotin has been shown to occur in <100 milliseconds.1 Biotin apparently blocks the interaction of the fluorophore with residues in the biotin-binding pocket that quench the fluorescence, enhancing the fluorescence of the stained tissue, often multifold. Figure. Spectra showing the fluorescence of 1) fluorescein-labeled avidin, 2) fluorescein-labeled avidin after addition of 10 µM biotin and 3) free fluorescein at the same concentration as the fluorescein label in the avidin conjugate [Image] Reference: 1. " Imaging of endosome fusion in BHK fibroblasts based on a novel fluorimetric avidin-biotin binding assay." Emans N, Biwersi J, Verkman AS. Biophys J 69, 716-728 (1995) Robert Archer, Ph.D. Cell Biology Product Manager mailto:robert@probes.com Molecular Probes, Inc. 4849 Pitchford Ave Eugene, OR 97402-9165 Tel: (541) 465-8353 Fax: (541) 465-4593 http://www.probes.com
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