critical comparison between two classical and a kit-based method for mitochondria isolation
Numerous protocols for isolation of mitochondria are available. Here, three methods for the isolation of intact mitochondria from mouse liver tissues are compared with regard to yield, purity and activity. Mitochondria were isolated by sucrose density gradient ultracentrifugation, free-flow electrop...
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| Published in: | Proteomics (Weinheim) Vol. 9; no. 11; pp. 3209 - 3214 |
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| Main Authors: | , , , , , , |
| Format: | Journal Article |
| Language: | English |
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Weinheim
Wiley-VCH Verlag
01-06-2009
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| Abstract | Numerous protocols for isolation of mitochondria are available. Here, three methods for the isolation of intact mitochondria from mouse liver tissues are compared with regard to yield, purity and activity. Mitochondria were isolated by sucrose density gradient ultracentrifugation, free-flow electrophoresis or a commercially available kit-based method. Our analyses show that the sophisticated (and most expensive) free-flow electrophoresis method enables isolation of intact mitochondria with an enrichment of approximately 70%. Using the classical density centrifugation method is very laborious and time-consuming, but delivers about 57% intact mitochondria. Using standard laboratory equipment in a quick and simple procedure, the kit provides approximately 50% intact mitochondria, suitable for most standard investigations. |
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| AbstractList | Numerous protocols for isolation of mitochondria are available. Here, three methods for the isolation of intact mitochondria from mouse liver tissues are compared with regard to yield, purity and activity. Mitochondria were isolated by sucrose density gradient ultracentrifugation, free‐flow electrophoresis or a commercially available kit‐based method. Our analyses show that the sophisticated (and most expensive) free‐flow electrophoresis method enables isolation of intact mitochondria with an enrichment of approximately 70%. Using the classical density centrifugation method is very laborious and time‐consuming, but delivers about 57% intact mitochondria. Using standard laboratory equipment in a quick and simple procedure, the kit provides approximately 50% intact mitochondria, suitable for most standard investigations. |
| Author | Feckler, Christian Lehr, Stefan Wolgast, Heike Müller-Wieland, Dirk Kotzka, Jörg Hartwig, Sonja Wallbrecht, Katrin |
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| Cites_doi | 10.1002/elps.1150180519 10.1002/elps.200305834 10.1042/bj0800649 10.1016/S0076-6879(84)05016-3 10.1002/pmic.200300376 10.1074/mcp.M800109-MCP200 10.1016/S0021-9258(17)42627-5 10.1002/1615-9861(200203)2:3<280::AID-PROT280>3.0.CO;2-W 10.1126/science.283.5407.1482 10.1021/bi0479656 10.1002/0471143030.cb0304s04 10.1046/j.1432-1327.1999.00725.x |
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| References_xml | – volume: 264 start-page: 687 year: 1999 end-page: 701 article-title: Mitochondria and cell death. Mechanistic and methodological issues publication-title: Eur. J. Biochem. – volume: 3 start-page: 906 year: 2003 end-page: 916 article-title: Improved proteome analysis of mitochondria by free‐flow electrophoresis publication-title: Proteomics – volume: 25 start-page: 1735 year: 2004 end-page: 1747 article-title: Efficient separation and analysis of peroxisomal membrane proteins using free‐flow isoelectric focusing publication-title: Electrophoresis – year: 2001 article-title: Purification of a crude mitochondrial fraction by density‐gradient centrifugation publication-title: Curr. Protoc. Cell Biol. – volume: 7 start-page: 1737 year: 2008 end-page: 1747 article-title: A comparative study of early liver dysfunction in senescence‐accelerated mouse using mitochondrial proteomics approaches publication-title: Mol Cell Proteomics – volume: 18 start-page: 774 year: 1997 end-page: 780 article-title: Isolation of rat hepatic peroxisomes by means of immune free flow electrophoresis publication-title: Electrophoresis – volume: 259 start-page: 14498 year: 1984 end-page: 14504 article-title: Developmental profiles and properties of hepatic peroxisomal apo‐ and mitochondrial holoalanine:glyoxylate aminotransferase during chick embryogenesis publication-title: J. Biol. Chem. – volume: 64 start-page: 61 year: 2006 end-page: 71 article-title: Overview on visceral manifestations of mitochondrial disorders publication-title: Neth. J. Med. – volume: 283 start-page: 1482 year: 1999 end-page: 1488 article-title: Mitochondrial diseases in man and mouse publication-title: Science – volume: 105 start-page: 121 year: 1984 end-page: 126 article-title: Catalase publication-title: Methods Enzymol. – year: 1974 – volume: 44 start-page: 5117 year: 2005 end-page: 5128 article-title: Effect of sterol regulatory element binding protein‐1a on the mitochondrial protein pattern in human liver cells detected by 2D‐DIGE publication-title: Biochemistry – volume: 80 start-page: 649 year: 1961 end-page: 654 article-title: Biochemistry of dystrophic muscle. Mitochondrial succinate‐tetrazolium reductase and adenosine triphosphatase publication-title: Biochem. J. – volume: 2 start-page: 280 year: 2002 end-page: 287 article-title: Primary skin fibroblasts as human model system for proteome analysis publication-title: Proteomics – ident: e_1_2_3_15_2 doi: 10.1002/elps.1150180519 – ident: e_1_2_3_14_2 doi: 10.1002/elps.200305834 – ident: e_1_2_3_5_2 doi: 10.1042/bj0800649 – ident: e_1_2_3_7_2 doi: 10.1016/S0076-6879(84)05016-3 – ident: e_1_2_3_12_2 doi: 10.1002/pmic.200300376 – ident: e_1_2_3_10_2 doi: 10.1074/mcp.M800109-MCP200 – volume-title: Methoden der Enzymatischen Analyse year: 1974 ident: e_1_2_3_6_2 contributor: fullname: Bergmeyer H. U. – volume: 259 start-page: 14498 year: 1984 ident: e_1_2_3_9_2 article-title: Developmental profiles and properties of hepatic peroxisomal apo‐ and mitochondrial holoalanine:glyoxylate aminotransferase during chick embryogenesis publication-title: J. Biol. Chem. doi: 10.1016/S0021-9258(17)42627-5 contributor: fullname: Noguchi T. – ident: e_1_2_3_13_2 doi: 10.1002/1615-9861(200203)2:3<280::AID-PROT280>3.0.CO;2-W – ident: e_1_2_3_2_2 doi: 10.1126/science.283.5407.1482 – ident: e_1_2_3_11_2 doi: 10.1021/bi0479656 – ident: e_1_2_3_8_2 doi: 10.1002/0471143030.cb0304s04 – volume: 64 start-page: 61 year: 2006 ident: e_1_2_3_3_2 article-title: Overview on visceral manifestations of mitochondrial disorders publication-title: Neth. J. Med. contributor: fullname: Finsterer J. – volume: 264 start-page: 687 year: 1999 ident: e_1_2_3_4_2 article-title: Mitochondria and cell death. Mechanistic and methodological issues publication-title: Eur. J. Biochem. doi: 10.1046/j.1432-1327.1999.00725.x contributor: fullname: Bernardi P. |
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| SubjectTerms | 2-D gels Analytical, structural and metabolic biochemistry Animals Biological and medical sciences Blotting, Western Cell Fractionation - methods Electrophoresis - methods Electrophoresis, Gel, Two-Dimensional Fundamental and applied biological sciences. Psychology Mice Miscellaneous Mitochondria Mitochondria, Liver Proteins Proteomics Proteomics methods Reproducibility of Results Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Subcellular fractionation Succinate Dehydrogenase - metabolism Ultracentrifugation - methods |
| Title | critical comparison between two classical and a kit-based method for mitochondria isolation |
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