Visualization and Purification of Germ Granule Proteins Using Proximity Labeling.

Membraneless organelles, such as germ granules and stress granules, are liquid-like condensates formed by phase transition. Recently, we and others have adopted proximity-based labeling methods to determine the composition of these membraneless compartments. Here, we describe the use of TurboID-an engineered promiscuous biotin ligase-to label and purify proteins localizing to germ granules, known as P granules. We provide a detailed protocol for visualization of the subcellular localization of biotinylated proteins from dissected gonads, assessment of TurboID enrichment using streptavidin blots, and enrichment of biotinylated proteins under stringent conditions. Altogether, this protocol provides a workflow to unravel the proteome of germ granules. Importantly, the assays described here can be applied to interrogate many membraneless organelles, in a diversity of living multicellular organisms. Graphical abstract.