Monitoring 14-3-3 protein interactions with a homogeneous, fluorescence polarization assay
Monitoring 14-3-3 protein interactions with a homogeneous, fluorescence polarization assay
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DOI：
10.1177/1087057105284862
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发表时间：
2006-04-01
影响因子：
--
通讯作者：
Fu, H
中科院分区：
文献类型：
--
作者：
Du, YH;Masters, SC;Fu, H
14-3-3蛋白介导磷酸化依赖性蛋白-蛋白相互作用。14-3-3通过与许多客户蛋白结合，控制着广泛的生理过程，并与多种疾病有关，包括癌症和神经退行性疾病。为了更好地了解14-3-3蛋白的结构和功能，开发14-3-3蛋白的小分子调节剂用于生理研究和潜在的治疗干预，作者设计并优化了一种高灵敏度的基于荧光偏振（FP）的14-3-3检测方法。利用14-3-3与来自Raf- I模型系统的荧光标记磷酸肽的相互作用，他们实现了一种简单的I步“illixand - I -测量”方法来分析14-3-3蛋白。这是一种基于溶液的通用方法，可用于监测14-3-3与各种客户蛋白的结合。14-3-3 FP分析方法非常稳定，在384口井中实现了稳健的性能，信噪比大于10，Z′因子大于0.7。由于其简单和高灵敏度，该方法通常适用于研究14-3-3/客户蛋白相互作用，特别适用于14-3-3调节剂的高通量筛选。
The 14-3-3 proteins mediate phosphorylation-dependent protein-protein interactions. Through binding to numerous client proteins, 14-3-3 controls a wide range of physiological processes and has been implicated in a variety of diseases, including cancer and neurodegenerative disorders. To better understand the Structure and function of 14-3-3 proteins and to develop small-molecule modulators of 14-3-3 proteins for physiological studies and potential therapeutic interventions, the authors have designed and optimized a highly sensitive fluorescence polarization (FP)-based 14-3-3 assay. Using the interaction of 14-3-3 with a fluorescently labeled phosphopeptide frorn Raf- I asa model system, they have achieved a simple I -step "Illixand-i-neasure" method for analyzing 14-3-3 proteins. This is a solution-based, versatile method that can be used to monitor the binding of 14-3-3 with a variety of client proteins. The 14-3-3 FP assay is highly stable and has achieved a robust performance in a 384-well format with a demonstrated signal-to-noise ratio greater than 10 and a Z' factor greater than 0.7. Because of its simplicity and high sensitivity, this assay is generally applicable to studying 14-3-3/client-protein interactions and especially valuable for high-throughput screening of 14-3-3 modulators.