Analysis of protein-ligand interactions by fluorescence polarization.
Analysis of protein-ligand interactions by fluorescence polarization.
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DOI：
10.1038/nprot.2011.305
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发表时间：
2011-03
期刊：
影响因子：
14.8
通讯作者：
中科院分区：
文献类型：
--
作者：
量化生物分子之间的关联是预测和理解支撑所有生物活性的相互作用所必需的。荧光偏振（FP）提供了一种非破坏性的方法来测量荧光配体与大分子的结合。我们描述了一种荧光素标记的肌醇1,4,5-三磷酸（IP3）与IP3受体n端片段的结合可以在不同的温度下和与其他配体的竞争中表征。测定配体结合的标准吉布斯自由能（ΔG°）、焓（ΔH°）和熵（ΔS°）变化。该方法适用于任何可获得适当荧光配体的纯化配体结合位点。FP可以在不使用放射性物质的情况下实时测量低亲和相互作用，它是非破坏性的，并且在适当的注意下，它可以分辨ΔH°和ΔS°。方案的第一部分，蛋白质制备，可能需要几个星期，而FP测量，一旦他们被优化，通常需要1-6小时。
Quantification of the associations between biomolecules is required both to predict and understand the interactions that underpin all biological activity. Fluorescence polarization (FP) provides a non-disruptive means of measuring the association of a fluorescent ligand with a larger molecule. We describe an FP assay in which binding of fluorescein-labelled inositol 1,4,5-trisphosphate (IP3) to N-terminal fragments of IP3 receptors can be characterised at different temperatures and in competition with other ligands. The assay allows the standard Gibbs free energy (ΔG°), enthalpy (ΔH°) and entropy (ΔS°) changes of ligand binding to be determined. The method is applicable to any purified ligand-binding site for which an appropriate fluorescent ligand is available. FP can be used to measure low-affinity interactions in real-time without use of radioactive materials, it is non-destructive, and with appropriate care it can resolve ΔH° and ΔS°. The first part of the protocol, protein preparation, may take several weeks, while the FP measurements, once they have been optimised, would normally take 1-6 h.
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