Isolation and analysis of fractions enriched in WrappER-associated mitochondria from mouse liver

• Published in: STAR protocols Vol. 2; no. 3; p. 100752
• Main Authors: Ilacqua, Nicolò, Anastasia, Irene, Pellegrini, Luca
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 17.09.2021; Elsevier
• Subjects: Animals; Cell Biology; Cell separation/fractionation; Endoplasmic Reticulum; Liver - chemistry; Liver - cytology; Metabolism; Mice; Microscopy, Electron; Mitochondria, Liver; Molecular Biology - instrumentation; Molecular Biology - methods; Protein Biochemistry; Proteomics - methods; Protocol; Cell separation/fractionation; Protein Biochemistry; Metabolism; Cell Biology
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2021.100752

The endoplasmic reticulum (ER) plays a central role in lipid homeostasis, but the role of individual ER subdomains in lipid biology has not been elucidated. WrappER is a curved wrapping type of rough-ER that establishes extensive contacts with almost every mitochondria of the hepatocyte in the mouse liver. Here, we describe a protocol for isolation of fractions enriched in wrappER-associated mitochondria from the mouse liver. We also provide techniques for assessing its quality by electron microscopy and biochemical/proteomic analysis. For complete information on the use and execution of this protocol, please refer to Anastasia et al. (2021). [Display omitted] •Protocol for isolating fractions enriched in wrappER-associated mitochondria (WAM)•Companion protocol for isolating a control ER-enriched fraction•Procedure to validate WAM- and ER-enriched fractions by electron microscopy analysis•Procedure to validate WAM- and ER-enriched fractions by immunoblot analysis The endoplasmic reticulum (ER) plays a central role in lipid homeostasis, but the role of individual ER subdomains in lipid biology has not been elucidated. WrappER is a curved wrapping type of rough-ER that establishes extensive contacts with almost every mitochondria of the hepatocyte in the mouse liver. Here, we describe a protocol for isolation of fractions enriched in wrappER-associated mitochondria from the mouse liver. We also provide techniques for assessing its quality by electron microscopy and biochemical/proteomic analysis.