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Updated: Aug 20, 2026
In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions
Published on: July 17, 2019
Electrophoretic behavior of streptavidin complexed to a biotinylated probe: a functional screening assay for
Nicolas Humbert1, Andrea Zocchi, Thomas R Ward
1Institute of Chemistry, University of Neuchâtel, Neuchâtel, Switzerland.
Abstract:
The biotin-binding protein streptavidin exhibits a high stability against thermal denaturation, especially when complexed to biotin. Herein we show that, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), streptavidin is stabilized at high temperature in the presence of biotinylated fluorescent probes, such as biotin-4-fluorescein, which is incorporated within the binding pocket. In nondenaturing SDS-PAGE, streptavidin is detectable when complexed with biotin-4-fluorescein using a UV-transilluminator. Using biotin-4-fluorescein, the detection limit of streptavidin lies in the same range as with Coomassie blue staining. The functionality of streptavidin mutants can readily be assessed from crude bacterial extracts using biotin-4-fluorescein as a probe in nondenaturing SDS-PAGE.
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