Electrophoretic behavior of streptavidin complexed to a biotinylated probe: a functional screening assay for biotin-binding proteins.
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.
Humbert
Nicolas
N
Institute of Chemistry, University of Neuchâtel, Neuchâtel, Switzerland.
Zocchi
Andrea
A
Ward
Thomas R
TR
D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D005452:Fluoresceins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D009154:Mutation / Q000235:genetics; D019809:Streptavidin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*
D005452:Fluoresceins; D005456:Fluorescent Dyes; C118194:biotin-4-fluorescein; D001710:Biotin; D019809:Streptavidin