Observing Real-Time Ubiquitination in High Throughput with Fluorescence Polarization.
Observing Real-Time Ubiquitination in High Throughput with Fluorescence Polarization.
复制标题
通过荧光偏振观察高通量实时泛素化。
DOI：
10.1007/978-1-0716-2784-6_1
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Pruneda,JonathanN
中科院分区：
文献类型：
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作者：
Franklin,TylerG;Pruneda,JonathanN
泛素结合和解结合的体外重建提供了获得酶动力学，特异性和结构-功能关系的有价值的信息。传统上，这些生化测定最终通过SDS-PAGE进行分离并通过免疫印迹进行分析，这是一种需要额外时间的方法，可能难以定量，并提供反应进程的颗粒快照。为了解决这些限制，我们已经实施了基于荧光偏振的测定，其基于分子量的变化真实的实时跟踪泛素缀合和解缀合。我们发现这种方法，我们称之为“UbiReal”，大大方便了生化研究，如突变分析，特异性测定和抑制剂表征。
Reconstitution of ubiquitin conjugation and deconjugation in vitro provides access to valuable information on enzyme kinetics, specificity, and structure–function relationships. Classically, these biochemical assays culminate in separation by SDS-PAGE and analysis by immunoblotting, an approach that requires additional time, can be difficult to quantify, and provides granular snapshots of the reaction progression. To address these limitations, we have implemented a fluorescence polarization-based assay that tracks ubiquitin conjugation and deconjugation in real time based upon changes in molecular weight. We find this approach, which we have termed “UbiReal,” to greatly facilitate biochemical studies such as mutational analyses, specificity determination, and inhibitor characterization.