title: A micropreparation of mitochondria from cells using magnetic beads with immunoaffinity.
abstract: Mitochondrial preparation is a key technique in the study of mitochondria. Growing evidence has demonstrated that mitochondrial proteins are tissue or cell type dependent. Locating the proteins in the global presence of mitochondrial membranes is a primary consideration in adopting antibodies for affinity enrichment of mitochondria on a micro scale. Two proteins located on the outer membrane of mitochondria, cytochrome b5 type B (CYB5B) and synaptojanin-2-binding protein (SYNJ2BP), were selected as candidates based on a survey of databases and the literature. The polyclonal antibodies against the truncated CYB5B and SYNJ2BP exhibited specific recognition to mitochondria and wider sensitivity to several tested mouse tissues and cell lines, whereas the antibody 22-kDa translocase of the outer mitochondrial membrane (TOM22) nearly missed detection of mitochondria in the liver and responded minimally to mitochondria from H9C2 and L-02 cells. Through the affinity enrichment for cellular mitochondria using magnetic beads coated with anti-CYB5B or anti-SYNJ2BP, we found that the anti-CYB5B beads could enrich mitochondria more efficiently even on a scale of 10,000 cultured cells. For the integrity and protein components, the enriched mitochondria on anti-CYB5B were carefully examined and were accepted in further functional study. We propose that an anti-CYB5B immunomagnetic approach is feasible in the micropreparation of mitochondria from cultured cells.
authors.affiliation: Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 101318, China.
authors.forename: Yawei
authors.initials: Y
authors.lastname: Ru
authors.forename: Liang
authors.initials: L
authors.lastname: Yin
authors.forename: Haidan
authors.initials: H
authors.lastname: Sun
authors.forename: Songyue
authors.initials: S
authors.lastname: Yin
authors.forename: Qin
authors.initials: Q
authors.lastname: Pan
authors.forename: Hanfu
authors.initials: H
authors.lastname: Wei
authors.forename: Lin
authors.initials: L
authors.lastname: Wu
authors.forename: Siqi
authors.initials: S
authors.lastname: Liu
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000276:immunology; D000818:Animals; D000915:Antibody Affinity; D001483:Base Sequence; D002458:Cell Fractionation / Q000379:methods* / Q000592:standards; D002460:Cell Line; D015786:Cytochromes b5 / Q000235:genetics / Q000276:immunology; D017931:DNA Primers / Q000235:genetics; D018189:Immunomagnetic Separation / Q000379:methods* / Q000592:standards; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology; D051379:Mice; D008928:Mitochondria / Q000737:chemistry* / Q000276:immunology*; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology; D040901:Proteomics / Q000379:methods; D011786:Quality Control; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C571539:CYB5B protein, mouse; D017931:DNA Primers; D008565:Membrane Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C457224:Synj2bp protein, mouse; D015786:Cytochromes b5