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首页> 外文期刊>Current Protocols in Protein Science >A Fluorescence-Based Assay to Monitor SUMOylation in Real-Time
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A Fluorescence-Based Assay to Monitor SUMOylation in Real-Time

机译：监控SUMOylation荧光技术分析在实时

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摘要

The small ubiquitin-like modifier (SUMO) is an important post-translational modifier that regulates various cellular processes. Extensive investigations have been made to comprehend the enzymatic process and consequence of SUMOylation. In vitro SUMOylation assays are invaluable for understanding the fundamental mechanisms of SUMOylation. A majority of these assays monitor changes in the size of the substrate upon SUMO conjugation. Current methods typically detect the size difference through SDS-PAGEand western blots, which makes these methods cumbersome, error-prone, and time-consuming. Here, we describe a fluorescence-based assay for real-time detection of SUMOylation. In the method, a fluorophore-tagged substrate is used in the SUMOylation reaction. Upon SUMOylation, the size and correlation time (tc) of the substrate increases, and so does its anisotropy. The rate of change in anisotropy with time reflects the efficiency of the SUMOylation machinery. The real-time SUMOylation assay protocol iselegant, time-saving, and less prone to errors.
机译：小ubiquitin-like修饰符(相扑)是一个重要的翻译后修饰符调节各种细胞过程。调查了理解酶SUMOylation的过程和结果。体外SUMOylation化验是无价的理解的基本机制SUMOylation。底物在相扑的大小的变化接合。通过SDS-PAGEand西方大小差异的屁股,这使得这些方法繁琐,容易出错,而且耗时。描述一个实时荧光技术分析SUMOylation检测。fluorophore-tagged衬底中使用SUMOylation反应。和关联时间(tc)的基质增加,那么它的各向异性。各向异性随时间的变化反映了SUMOylation机械效率。实时SUMOylation iselegant试验协议,节省时间,也不太容易出错。

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