Fluorometric Assay for Quantitation of Biotin Covalently Attached to Proteins and Nucleic Acids

Abstract

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 Fluor® 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.

AIM2 Inflammasome-Mediated Pyroptosis in Enterovirus A71-Infected Neuronal Cells Restricts Viral Replication

“…Cell pellets were fixed with Fluorofix buffer (Biolegend, USA) for 30 min at room temperature (RT) in the dark 87–89 , and PBS-washed twice before permeabilization with 0.005% Triton-X100 for 10 min. Cells were then incubated for 1 hr at RT with mouse monoclonal Enterovirus blend 3321 antibody (dilution 1:50; Merck, Germany), followed by goat-anti mouse IgG conjugated with Alexa-fluor 488 (Molecular Probes, USA) for 30 min at RT in the dark 90 . Cells were re-suspended in cell staining buffer (Biolegend, USA) before analysis using the FACS Canto II Flow cytometer (Becton Dickson, USA) and the Flowjo, Single Cell Analysis Software version 10 (FlowJo,USA).…”

Characterization of Contrast Agent Microbubbles for Ultrasound Imaging and Therapy Research

“…Qualitative determination of incorporation into a microbubble shell can be achieved by widely available fluorescent biotin or avidin. Quantitatively, colorimetric and fluorimetric tests using 2-(4 -hydroxyazobenzene) benzoic acid (HABA) are available to rapidly determine biotin levels in a sample [130], [131]. This has been performed for biotinylated albumin to determine the binding efficiency of the conjugation of biotin to albumin subsequently used to create microbubbles [132] and on antibodies to be conjugated to microbubbles via avidin-biotin [133].…”

Amphipol-Mediated Screening of Molecular Orthoses Specific for Membrane Protein Targets

“…1b), the average molecular mass, is *14 kDa, and the number-average degree of polymerization is *20. The extent of grafting of the biotin group was estimated to be *0.56 per polymer chain by means of a combination of 1 H NMR and spectrophotometric test using 4-hydroxyazobenzene-2-carboxylic acid, an avidin-binding dye that can be stoichiometrically displaced by biotin (Batchelor et al 2007). …”