内源性非锚定多聚泛素链的纯化方法。
Method for the Purification of Endogenous Unanchored Polyubiquitin Chains.
作者信息
Scott Daniel, Strachan Jo, Krishna Varun Gopala, Shaw Barry, Tooth David J, Searle Mark S, Oldham Neil J, Layfield Rob
机构信息
School of Life Sciences, Queen's Medical Centre, University of Nottingham, Nottingham, NG7 2UH, UK.
Institute of Cell Biology, University of Edinburgh, Edinburgh, UK.
出版信息
Methods Mol Biol. 2016;1449:203-13. doi: 10.1007/978-1-4939-3756-1_11.
Unanchored polyubiquitin chains are endogenous non-substrate linked ubiquitin polymers which have emerging roles in the control of cellular physiology. We describe an affinity purification method based on an isolated ubiquitin-binding domain, the ZnF_UBP domain of the deubiquitinating enzyme USP5, which permits the selective purification of mixtures of endogenous unanchored polyubiquitin chains that are amenable to downstream molecular analyses. Further, we present methods for detection of unanchored polyubiquitin chains in purified fractions.
非锚定多聚泛素链是内源性非底物连接的泛素聚合物，在细胞生理学调控中发挥着越来越重要的作用。我们描述了一种基于分离的泛素结合结构域（去泛素化酶USP5的ZnF_UBP结构域）的亲和纯化方法，该方法能够选择性地纯化内源性非锚定多聚泛素链混合物，便于进行下游分子分析。此外，我们还介绍了检测纯化组分中非锚定多聚泛素链的方法。
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