Batchelor Robert H, Sarkez Adam, Cox W Gregory, Johnson Iain
Molecular Probes Detection Technologies, Invitrogen, Eugene, OR 97402, USA.
Biotechniques. 2007 Oct;43(4):503-7. doi: 10.2144/000112564.
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.
作为（链霉）抗生物素蛋白亲和系统的一个组成部分，生物素常常与蛋白质或核酸共价连接。我们在此描述一种基于微孔板的高通量荧光测定法，用于检测与蛋白质或核酸相连的生物素，该方法基于荧光共振能量转移（FRET）。此测定法利用了一种复合物，即Alexa Fluoro 488染料标记的抗生物素蛋白与一种猝灭染料2-（4'-羟基偶氮苯）苯甲酸（HABA）形成的复合物，其中HABA占据了抗生物素蛋白的生物素结合位点。在没有生物素的情况下，HABA通过FRET猝灭Alexa Fluoro 488染料的荧光发射。当生物素与Alexa Fluoro 488染料标记的抗生物素蛋白结合时，HABA被取代，导致FRET效率降低。这种机制使得荧光强度增加，且荧光强度的增加与样品中生物素的含量直接相关。该测定法在将样品加入试剂后15分钟内，能够在0.1 mL体积中检测到低至4 pmol的生物素，Z因子> 0.9。
