Protocol for the biochemical isolation of endosomal membranes associated with endoplasmic reticulum in HeLa cells

Endosome-endoplasmic reticulum contact sites (EERCS) are highly dynamic membrane interfaces that regulate trafficking and signaling, yet biochemical approaches to isolate them remain nonexistent, unlike established protocols for other types of contacts. Here, we present a protocol to isolate and purify endosomal membranes associated with endoplasmic reticulum membranes from cultured mammalian cells using subcellular fractionation and a two-step density-gradient centrifugation. We describe steps for cell expansion, post-nuclear supernatant collection, and the isolation of light membranes and EERCS. For complete details on the use and execution of this protocol, please refer to Da Graça et al.¹.