A HABA dye-based colorimetric assay to detect unoccupied biotin binding sites in an avidin-containing fusion protein

Avidin-biotin binding, the most robust non-covalent protein-ligand interaction occurring in nature, has wide-ranging applications in biotechnology. A frequent challenge in these applications is accurately determining the number of unoccupied biotin binding sites in avidin-containing fusion proteins....

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Veröffentlicht in:BioTechniques. - 1991. - 76(2024), 10 vom: 07. Okt., Seite 485-494
1. Verfasser: Mukherjee, Sonia (VerfasserIn)
Weitere Verfasser: Leblanc, Pierre, Poznansky, Mark C, Sluder, Ann E
Format: Online-Aufsatz
Sprache:English
Veröffentlicht: 2024
Zugriff auf das übergeordnete Werk:BioTechniques
Schlagworte:Journal Article avidin binding assay binding site occupancy biotin fusion protein self-assembling vaccine Biotin 6SO6U10H04 Avidin mehr... 1405-69-2 Recombinant Fusion Proteins Coloring Agents Azo Compounds
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520 |a Avidin-biotin binding, the most robust non-covalent protein-ligand interaction occurring in nature, has wide-ranging applications in biotechnology. A frequent challenge in these applications is accurately determining the number of unoccupied biotin binding sites in avidin-containing fusion proteins. We delineate a novel assay protocol in miniaturized format to quantify available biotin binding sites based on the affinity of the anionic dye 4'-hydroxyazobenzene-2-carboxylic acid for biotin binding sites within avidin. We apply this assay as a quality control assay to evaluate the number of available biotin binding sites in different fusion protein production batches. This method offers a streamlined alternative to fluorescence-based assays commonly employed to assess biotin binding, is less time-consuming than other methods and is applicable to diverse fusion proteins 
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700 1 |a Leblanc, Pierre  |e verfasserin  |4 aut 
700 1 |a Poznansky, Mark C  |e verfasserin  |4 aut 
700 1 |a Sluder, Ann E  |e verfasserin  |4 aut 
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