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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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2024-10-01
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| Series: | BioTechniques |
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| Online Access: | https://www.tandfonline.com/doi/10.1080/07366205.2024.2397288 |
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| author | Sonia Mukherjee Pierre Leblanc Mark C Poznansky Ann E Sluder |
| author_facet | Sonia Mukherjee Pierre Leblanc Mark C Poznansky Ann E Sluder |
| author_sort | Sonia Mukherjee |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-c3a988544e4e41a998d75a6da60b4f19 |
| institution | Kabale University |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2024-10-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-c3a988544e4e41a998d75a6da60b4f192025-01-16T15:42:49ZengTaylor & Francis GroupBioTechniques0736-62051940-98182024-10-01761048549410.1080/07366205.2024.2397288A HABA dye-based colorimetric assay to detect unoccupied biotin binding sites in an avidin-containing fusion proteinSonia Mukherjee0Pierre Leblanc1Mark C Poznansky2Ann E Sluder3Vaccine & Immunotherapy Center, Massachusetts General Hospital, Boston, MA 02114, USAVaccine & Immunotherapy Center, Massachusetts General Hospital, Boston, MA 02114, USAVaccine & Immunotherapy Center, Massachusetts General Hospital, Boston, MA 02114, USAVaccine & Immunotherapy Center, Massachusetts General Hospital, Boston, MA 02114, USAAvidin-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.https://www.tandfonline.com/doi/10.1080/07366205.2024.2397288avidinbinding assaybinding site occupancybiotinfusion proteinself-assembling vaccine |
| spellingShingle | Sonia Mukherjee Pierre Leblanc Mark C Poznansky Ann E Sluder A HABA dye-based colorimetric assay to detect unoccupied biotin binding sites in an avidin-containing fusion protein BioTechniques avidin binding assay binding site occupancy biotin fusion protein self-assembling vaccine |
| title | A HABA dye-based colorimetric assay to detect unoccupied biotin binding sites in an avidin-containing fusion protein |
| title_full | A HABA dye-based colorimetric assay to detect unoccupied biotin binding sites in an avidin-containing fusion protein |
| title_fullStr | A HABA dye-based colorimetric assay to detect unoccupied biotin binding sites in an avidin-containing fusion protein |
| title_full_unstemmed | A HABA dye-based colorimetric assay to detect unoccupied biotin binding sites in an avidin-containing fusion protein |
| title_short | A HABA dye-based colorimetric assay to detect unoccupied biotin binding sites in an avidin-containing fusion protein |
| title_sort | haba dye based colorimetric assay to detect unoccupied biotin binding sites in an avidin containing fusion protein |
| topic | avidin binding assay binding site occupancy biotin fusion protein self-assembling vaccine |
| url | https://www.tandfonline.com/doi/10.1080/07366205.2024.2397288 |
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