title: Isolation of functional pure mitochondria by superparamagnetic microbeads.
abstract: Isolation of mitochondria by current methods relies mainly on their physicochemical properties. Here we describe an alternative approach to obtain functional mitochondria from human cells in a fast, reproducible, and standardized procedure. The new approach is based on superparamagnetic microbeads conjugated to anti-TOM22 antibody. The bead conjugates label the cytoplasmic part of the human mitochondrial membrane protein TOM22 and, thus, allow for a gentle isolation of mitochondria in a high gradient magnetic field. By comparing the MACS (magnetic cell separation) approach with mitochondria isolation methods using differential centrifugation and ultracentrifugation we demonstrate that the MACS approach provides the highest yield of isolated mitochondria. The quality, enrichment, and purity of mitochondria isolated with this protocol are comparable to mitochondria obtained using the ultracentrifuge method, and a typical separation procedure takes only approximately 1 to 2h from initial cell homogenization. Mitochondria isolated with the new approach are sufficient for protein import, blue native gel electrophoresis, and other mitochondrial assays.
authors.affiliation: Institute of Vegetative Physiology, Medical Faculty, University of Köln, 50931 Köln, Germany. hue-tran.hornig@uni-koeln.de
authors.forename: Hue-Tran
authors.initials: HT
authors.lastname: Hornig-Do
authors.forename: Gritt
authors.initials: G
authors.lastname: Günther
authors.forename: Maria
authors.initials: M
authors.lastname: Bust
authors.forename: Patricia
authors.initials: P
authors.lastname: Lehnartz
authors.forename: Andreas
authors.initials: A
authors.lastname: Bosio
authors.forename: Rudolf J
authors.initials: RJ
authors.lastname: Wiesner
mesh_terms: D000906:Antibodies / Q000737:chemistry; D005434:Flow Cytometry; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods*; D008280:Magnetics; D008565:Membrane Proteins / Q000276:immunology; D008863:Microspheres; D008928:Mitochondria; D024101:Mitochondrial Proteins / Q000276:immunology
chemical_list: D000906:Antibodies; D008565:Membrane Proteins; D024101:Mitochondrial Proteins