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Here, we present a general protocol for mimicking lipid-mediated phase separation on the membrane using giant unilamellar vesicles (GUVs). In this protocol, we use GUVs to mimic Ago1 protein's phase separation behavior on the membrane through binding with phosphoinositides (PIPs). We provide procedures to prepare fluorescent-labeled Ago1 protein and PI(4,5)P2-containing GUVs, followed by steps to assess Ago1 protein's phase separation in 3D time-lapse images. This protocol can be applied to investigate a membrane-associated protein's behavior on the membrane. For complete details on the use and execution of this protocol, please refer to Gao et al. (2022).
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