Protocol for detecting protein recruitment to clathrin structures in cultured cells.

Clathrin-mediated endocytosis internalizes key cell-surface receptors and relies on coordinated recruitment of adaptors and accessory proteins to clathrin structures. Here, we present a protocol for quantifying protein recruitment to diffraction-limited clathrin structures at the plasma membrane. We describe steps for the inducible expression of tagged proteins, total internal reflection fluorescence (TIRF) microscopy, the analysis of proteins within clathrin structures, UltraID-based proximity-labeling, and western blotting detection of proteins in clathrin structures. This protocol is applicable to the detection of any protein within clathrin or related structures. For complete details on the use and execution of this protocol, please refer to Orofiamma et al.1.