High-throughput fluorescence polarization assay of ligand binding to IP3 receptors.
High-throughput fluorescence polarization assay of ligand binding to IP3 receptors.
复制标题
配体与 IP3 受体结合的高通量荧光偏振测定。
DOI：
10.1101/pdb.prot073080
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发表时间：
2013
影响因子：
--
通讯作者：
Rossi AM
中科院分区：
文献类型：
--
作者：
Rossi AM
荧光偏振（FP）允许定量小荧光配体与较大蛋白质的结合，因为游离配体比结合形式旋转得更快。该方案描述了在不同温度和竞争配体存在下荧光素标记的肌醇1，4，5-三磷酸（IP 3）与IP 3受体氨基末端片段结合的FP测定。该方法需要荧光素标记的IP 3和能够进行FP测量的读板器。该测定可以真实的时间测量低亲和力相互作用，其避免使用放射性材料，是非破坏性的，并且可以解析配体结合时发生的吉布斯自由能（ΔG）、焓（ΔH）和熵（ΔS）的变化。它适用于任何纯化的蛋白质，其荧光配体是可用的。优化后，该过程可在1-6 h内完成。
Fluorescence polarization (FP) allows quantification of the binding of a small fluorescent ligand to a larger protein because the free ligand rotates more rapidly than the bound form. This protocol describes an FP assay for the binding of fluorescein-labeled inositol 1, 4, 5-trisphosphate (IP 3) to amino-terminal fragments of the IP 3 receptor at different temperatures and in the presence of competing ligands. The method requires fluorescein-labeled IP 3 and a plate-reader capable of FP measurements. The assay can measure low-affinity interactions in real time, it avoids use of radioactive materials, is nondestructive, and can resolve changes in Gibbs free energy (ΔG), enthalpy (ΔH), and entropy (ΔS) that occur with ligand binding. It is applicable to any purified protein for which a fluorescent ligand is available. After optimization, the procedure can be completed in 1–6 h.