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A Simple Spectrophotometric Streptavidin-Biotin Binding Assay Utilizing Biotin-4-Fluorescein., Mark Waner, David Mascotti
A Simple Spectrophotometric Streptavidin-Biotin Binding Assay Utilizing Biotin-4-Fluorescein., Mark Waner, David Mascotti
David P. Mascotti
A new assay for biotin binding capacity of Streptavidin (SA) is presented in this work. The assay is based on the large decrease in the extinction coefficient at 493 nm that accompanies binding of biotin-4-fluorescein (B4F) to SA. This decrease is attributed to formation of a charge transfer complex between the B4F-donor and one or more SA residues. We show that one may observe the stoichiometric binding via monitoring the absorbance at 493 nm using either SA or B4F as the titrant. The sensitivity of the assay is at the lower end of similar fluorimetric and photometric assays. Though the …