A HABA dye-based colorimetric assay to detect unoccupied biotin binding sites in an avidin-containing fusion protein
| Autor: | Sonia Mukherjee, Pierre Leblanc, Mark C Poznansky, Ann E Sluder |
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| Jazyk: | angličtina |
| Rok vydání: | 2024 |
| Předmět: | |
| Zdroj: | BioTechniques, Pp 1-10 (2024) |
| Druh dokumentu: | article |
| ISSN: | 07366205 1940-9818 0736-6205 |
| DOI: | 10.1080/07366205.2024.2397288 |
| Popis: | 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. |
| Databáze: | Directory of Open Access Journals |
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