Mammalian MemPrep is an approach that enables the isolation of ER membranes from HEK293T cells at a purity suitable for quantitative proteomics and lipidomics. Using cleavable, epitope‑tagged variants of SEC61β and REEP5, which preferentially localize to ER sheets and tubules, respectively, we isolate membrane vesicles corresponding to the two major ER subdomains. The proteomic analysis confirms the high purity of our preparation and the expected distribution of protein machineries: sheet‑associated factors, including translocon components and ribosomal proteins, are enriched in SEC61β isolates, whereas curvature‑stabilizing proteins are enriched in REEP5 isolates.