Instituto de Bioquímica Vegetal y Fotosíntesis (IBVF), Universidad de Sevilla and Consejo Superior de Investigaciones Cientificas, Sevilla, Spain.
Methods Mol Biol. 2024;2832:57-66. doi: 10.1007/978-1-0716-3973-3_4.
Stress granules (SGs) are conserved cytoplasmic biomolecular condensates mainly formed by proteins and RNA molecules assembled by liquid-liquid phase separation. Isolation of SGs components has been a major challenge in the field due to the dynamic and transient nature of stress granule shells. Here, we describe the methodology for the isolation and visualization of SGs proteins from Arabidopsis thaliana plants using a scaffold component as the target. The protocol consists of the first immunoprecipitation of GFP-tagged scaffold protein, followed by an on-beads enzymatic digestion and previous mass spectrometry identification. Finally, the localization of selected SGs candidates is visualized in Nicotiana benthamiana mesophyll protoplasts.
应激颗粒（SGs）是由蛋白质和 RNA 分子组成的保守细胞质生物分子凝聚物，主要通过液-液相分离形成。由于应激颗粒壳的动态和瞬时性质，SGs 成分的分离一直是该领域的主要挑战。在这里，我们描述了一种使用支架成分作为靶标从拟南芥植物中分离和可视化 SGs 蛋白的方法。该方案包括 GFP 标记的支架蛋白的第一次免疫沉淀，然后进行珠上酶消化和先前的质谱鉴定。最后，在本氏烟叶肉原生质体中可视化选定的 SGs 候选物的定位。
