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Isolation of mitochondria from Saccharomyces cerevisiae using magnetic bead affinity purification

Isolated mitochondria are widely used to study the function of the organelle. Typically, mitochondria are prepared using differential centrifugation alone or in conjunction with density gradient ultracentrifugation. However, mitochondria isolated using differential centrifugation contain membrane or...

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Autores principales: Liao, Pin-Chao, Boldogh, Istvan R., Siegmund, Stephanie E., Freyberg, Zachary, Pon, Liza A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5919621/
https://www.ncbi.nlm.nih.gov/pubmed/29698455
http://dx.doi.org/10.1371/journal.pone.0196632
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author Liao, Pin-Chao
Boldogh, Istvan R.
Siegmund, Stephanie E.
Freyberg, Zachary
Pon, Liza A.
author_facet Liao, Pin-Chao
Boldogh, Istvan R.
Siegmund, Stephanie E.
Freyberg, Zachary
Pon, Liza A.
author_sort Liao, Pin-Chao
collection PubMed
description Isolated mitochondria are widely used to study the function of the organelle. Typically, mitochondria are prepared using differential centrifugation alone or in conjunction with density gradient ultracentrifugation. However, mitochondria isolated using differential centrifugation contain membrane or organelle contaminants, and further purification of crude mitochondria by density gradient ultracentrifugation requires large amounts of starting material, and is time-consuming. Mitochondria have also been isolated by irreversible binding to antibody-coated magnetic beads. We developed a method to prepare mitochondria from budding yeast that overcomes many of the limitations of other methods. Mitochondria are tagged by insertion of 6 histidines (6xHis) into the TOM70 (Translocase of outer membrane 70) gene at its chromosomal locus, isolated using Ni-NTA (nickel (II) nitrilotriacetic acid) paramagnetic beads and released from the magnetic beads by washing with imidazole. Mitochondria prepared using this method contain fewer contaminants, and are similar in ultrastructure as well as protein import and cytochrome c oxidase complex activity compared to mitochondria isolated by differential centrifugation. Moreover, this isolation method is amenable to small samples, faster than purification by differential and density gradient centrifugation, and more cost-effective than purification using antibody-coated magnetic beads. Importantly, this method can be applied to any cell type where the genetic modification can be introduced by CRISPR or other methods.
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spelling pubmed-59196212018-05-11 Isolation of mitochondria from Saccharomyces cerevisiae using magnetic bead affinity purification Liao, Pin-Chao Boldogh, Istvan R. Siegmund, Stephanie E. Freyberg, Zachary Pon, Liza A. PLoS One Research Article Isolated mitochondria are widely used to study the function of the organelle. Typically, mitochondria are prepared using differential centrifugation alone or in conjunction with density gradient ultracentrifugation. However, mitochondria isolated using differential centrifugation contain membrane or organelle contaminants, and further purification of crude mitochondria by density gradient ultracentrifugation requires large amounts of starting material, and is time-consuming. Mitochondria have also been isolated by irreversible binding to antibody-coated magnetic beads. We developed a method to prepare mitochondria from budding yeast that overcomes many of the limitations of other methods. Mitochondria are tagged by insertion of 6 histidines (6xHis) into the TOM70 (Translocase of outer membrane 70) gene at its chromosomal locus, isolated using Ni-NTA (nickel (II) nitrilotriacetic acid) paramagnetic beads and released from the magnetic beads by washing with imidazole. Mitochondria prepared using this method contain fewer contaminants, and are similar in ultrastructure as well as protein import and cytochrome c oxidase complex activity compared to mitochondria isolated by differential centrifugation. Moreover, this isolation method is amenable to small samples, faster than purification by differential and density gradient centrifugation, and more cost-effective than purification using antibody-coated magnetic beads. Importantly, this method can be applied to any cell type where the genetic modification can be introduced by CRISPR or other methods. Public Library of Science 2018-04-26 /pmc/articles/PMC5919621/ /pubmed/29698455 http://dx.doi.org/10.1371/journal.pone.0196632 Text en © 2018 Liao et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Liao, Pin-Chao
Boldogh, Istvan R.
Siegmund, Stephanie E.
Freyberg, Zachary
Pon, Liza A.
Isolation of mitochondria from Saccharomyces cerevisiae using magnetic bead affinity purification
title Isolation of mitochondria from Saccharomyces cerevisiae using magnetic bead affinity purification
title_full Isolation of mitochondria from Saccharomyces cerevisiae using magnetic bead affinity purification
title_fullStr Isolation of mitochondria from Saccharomyces cerevisiae using magnetic bead affinity purification
title_full_unstemmed Isolation of mitochondria from Saccharomyces cerevisiae using magnetic bead affinity purification
title_short Isolation of mitochondria from Saccharomyces cerevisiae using magnetic bead affinity purification
title_sort isolation of mitochondria from saccharomyces cerevisiae using magnetic bead affinity purification
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5919621/
https://www.ncbi.nlm.nih.gov/pubmed/29698455
http://dx.doi.org/10.1371/journal.pone.0196632
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