Fluorometric assay for quantitation of biotin covalently attached to proteins and nucleic acids
Fluorometric assay for quantitation of biotin covalently attached to proteins and nucleic acids
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DOI：
10.2144/000112564
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发表时间：
2007-10-01
期刊：
影响因子：
2.7
通讯作者：
Johnson, Iain
中科院分区：
文献类型：
--
作者：
Batchelor, Robert H.;Sarkez, Adam;Johnson, Iain
生物素作为链状亲和素亲和系统的一部分，通常与蛋白质或核酸共价连接。在这里，我们描述了一种基于荧光共振能量转移(FRET)的微孔板高通量荧光分析生物素与蛋白质或核酸的方法。这种攻击利用Alexa Fluor(R)488染料标记的亲和素与猝灭剂染料2-(4‘-羟基偶氮苯)苯甲酸(HABA)的络合物，占据亲和素的生物素结合部位。在没有生物素的情况下，HABA通过FRET猝灭Alexa Fluor 488染料的荧光发射，当生物素与Alexa Fluor 488染料标记的亲和素结合时，FRET被取代，导致FRET降低)。这一机制导致了荧光强度的增加，与生物素的含量直接相关。样本。在试剂中加入样品后15分钟内，该方法能够在0.1毫升体积内检测到低至4pmoL的生物素，Z因子为0.9。
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 assail utilizes a complex of Alexa Fluor (R) 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 e). 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.