一种用于调节 Rho GTPase 活性的基于荧光共振能量转移的筛选兼容活细胞荧光检测法。

A screening-compatible live cell fluorescence resonance energy transfer-based assay for modulation of Rho GTPase activity.

机构信息

Institute of Chemistry and Biochemistry, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany.

Institute of Biochemistry, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany.

出版信息

STAR Protoc. 2022 Dec 16;3(4):101705. doi: 10.1016/j.xpro.2022.101705. Epub 2022 Sep 22.

DOI:10.1016/j.xpro.2022.101705

PMID:36149795

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9508600/

Abstract

Rho family GTPases are central regulators of cytoskeletal dynamics controlled by guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs). This protocol presents a workflow for a robust high-throughput compatible biosensor assay to analyze changes in Rho GTPase activity by these proteins in the native cellular environment. The procedure can be used for semi-quantitative comparison of GEF/GAP function and extended for analysis of additional modulators. The experimental design is applicable also to other monomolecular ratiometric FRET sensors. For complete details on the use and execution of this protocol, please refer to Müller et al. (2020).

摘要

Rho 家族 GTPases 是细胞骨架动态的核心调节剂，由鸟嘌呤核苷酸交换因子（RhoGEFs）和 GTPase 激活蛋白（RhoGAPs）控制。本方案提供了一种适用于高通量兼容的生物传感器测定的稳健工作流程，用于分析这些蛋白在天然细胞环境中对 Rho GTPase 活性的变化。该程序可用于半定量比较 GEF/GAP 功能，并扩展到分析其他调节剂。实验设计也适用于其他单分子比率 FRET 传感器。如需了解本方案使用和执行的详细信息，请参阅 Müller 等人。(2020)。