title: Scalable Isolation of Mammalian Mitochondria for Nucleic Acid and Nucleoid Analysis.
abstract: Isolation of mitochondria from cultured cells and animal tissues for analysis of nucleic acids and bona fide mitochondrial nucleic acid binding proteins and enzymes is complicated by contamination with cellular nucleic acids and their adherent proteins. Protocols presented here allow for quick isolation of mitochondria from a small number of cells and for preparation of highly purified mitochondria from a larger number of cells using nuclease treatment and high salt washing of mitochondria to reduce contamination. We further describe a method for the isolation of mitochondrial DNA-protein complexes known as nucleoids from these highly purified mitochondria using a combination of glycerol gradient sedimentation followed by isopycnic centrifugation in a non-ionic iodixanol gradient.
authors.affiliation: Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY, 11794-8651, USA.|Memorial Sloan Kettering Cancer Institute, New York, NY, USA.
authors.forename: Ken-Wing
authors.initials: KW
authors.lastname: Lee
authors.affiliation: Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY, 11794-8651, USA. daniel.bogenhagen@stonybrook.edu.
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Bogenhagen
mesh_terms: D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics; D002499:Centrifugation, Density Gradient / Q000379:methods*; D002500:Centrifugation, Isopycnic / Q000379:methods*; D004272:DNA, Mitochondrial / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000302:isolation & purification; D006367:HeLa Cells; D006801:Humans; D008928:Mitochondria / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000378:metabolism; D000069396:Mitochondrial Ribosomes / Q000737:chemistry; D012313:RNA / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D000077278:RNA, Mitochondrial; D014283:Triiodobenzoic Acids / Q000737:chemistry
chemical_list: D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins; D024101:Mitochondrial Proteins; D000077278:RNA, Mitochondrial; D014283:Triiodobenzoic Acids; D012313:RNA; C044834:iodixanol
keywords: Mitochondria; Mitochondrial RNA; mtDNA nucleoids