利用分裂荧光素酶互补实验分析人体细胞裂解物中蛋白质与蛋白质之间相互作用的方法。

IF 1.3 Q4 BIOCHEMICAL RESEARCH METHODS

STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-10-04 DOI:10.1016/j.xpro.2024.103328

Zander Claes, Sarah Lemaire, Mathieu Bollen

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引用次数: 0

摘要

在这里，我们介绍了一种基于裂解物的分离荧光素酶测定法，用于检测 HEK293T 细胞裂解物中的蛋白质-蛋白质相互作用（PPIs），以蛋白磷酸酶 PP1 亚基之间的相互作用为例。我们介绍了存储和重复使用裂解物、传感器设计、检测设置/优化以及化合物库高通量筛选的步骤。然后，我们详细介绍了将该检测方法作为研究工具应用于表征 PPIs 动态的步骤，并通过具体实例进行了说明。有关使用和执行本方案的完整细节，请参阅 Claes 和 Bollen.1.

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Protocol for analyzing protein-protein interactions by split-luciferase complementation assays in human cell lysates.

Here, we present a lysate-based split-luciferase assay for examining protein-protein interactions (PPIs) in HEK293T cell lysates, exemplified by interactions between subunits of protein phosphatase PP1. We describe steps for storing and re-using lysates, sensor design, assay setup/optimization, and high-throughput screening of compound libraries. We then detail procedures for applying the assay as a research tool to characterize the dynamics of PPIs, which we illustrate with specific examples. For complete details on the use and execution of this protocol, please refer to Claes and Bollen.¹.

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