Electrophoretic behavior of streptavidin complexed to a biotinylated probe : A functional screening assay for biotin-binding proteins
Ward, Thomas R.
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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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Resource Types::text::journal::journal article
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