| Fluorometric assay for quantitation of biotin covalently attached to proteins and nucleic acids. | |
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MedLine Citation:
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PMID: 18019342 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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As a component of the (strept)avidin affinity system, biotin is often covalently linked to proteins or nucleic acids. We describe here a microplate-based high-throughput fluorometric assay for biotin linked to either proteins or nucleic acids based on fluorescence resonance energy transfer (FRET). This assay utilizes a complex of Alexa Fluoro 488 dye-labeled avidin with a quencher dye, 2-(4'-hydroxyazobenzene) benzoic acid (HABA), occupying the biotin binding sites of the avidin. In the absence of biotin, HABA quenches the fluorescence emission of the Alexa Fluor 488 dyes via FRET HABA is displaced when biotin binds to the Alexa Fluor 488 dye-labeled avidin, resulting in decreased FRET efficiency. This mechanism results in an increase in fluorescence intensity directly related to the amount of biotin present in the sample. The assay is able to detect as little as 4 pmol biotin in a 0.1 mL volume within 15 min of adding sample to the reagent, with a Z-factor > 0.9. |
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Authors:
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Robert H Batchelor; Adam Sarkez; W Gregory Cox; Iain Johnson |
Publication Detail:
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Type: Journal Article; Technical Report |
Journal Detail:
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Title: BioTechniques Volume: 43 ISSN: 0736-6205 ISO Abbreviation: BioTechniques Publication Date: 2007 Oct |
Date Detail:
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Created Date: 2007-11-16 Completed Date: 2007-12-13 Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 8306785 Medline TA: Biotechniques Country: England |
Other Details:
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Languages: eng Pagination: 503-7 Citation Subset: IM |
Affiliation:
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Molecular Probes Detection Technologies, Invitrogen, Eugene, OR 97402, USA. rob.batchelor@invitrogen.com |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Biotin
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metabolism* Fluorescence Resonance Energy Transfer / methods* Immunoassay / methods* Microarray Analysis / methods* Nucleic Acids / metabolism* Protein Binding Proteins / metabolism* |
| Chemical | |
Reg. No./Substance:
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0/Nucleic Acids; 0/Proteins; 58-85-5/Biotin |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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