Abstract：
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.
摘要：
应激颗粒（SGs）是保守的细胞质生物分子缩合物，主要由蛋白质和RNA分子通过液-液相分离组装而成。由于应力颗粒壳的动态和瞬态性质，SGs组件的隔离一直是该领域的主要挑战。这里,我们描述了使用支架组件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个免疫沉淀，然后进行珠子上的酶消化和先前的质谱鉴定。最后,所选择的SGs候选物的定位在Nicotianabethamiana叶肉原生质体中可视化。
