Mark Waner - A Simple Spectrophotometric Streptavidin-Biotin Binding Assay Utilizing Biotin-4-Fluorescein

Document created by Mark Waner on Aug 22, 2014
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  Publication Details (including relevant citation   information):

  Waner, M.J., Mascotti, D.P., J. Biochem. Biophys. Methods, 2008,   70, 873-877. (DOI 10.1016/j.jbbm.2007.06.001).


  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 sensitivity is not substantially lower than   other comparable techniques, this assay allows one added   flexibility in working range and instrumentation, since the same   stock solutions may be used for this new photometric assay or the   fluorescence assay for which this ligand was first developed.

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