Details
| Original language | English |
|---|---|
| Title of host publication | Methods in Molecular Biology |
| Pages | 121-133 |
| Number of pages | 13 |
| ISBN (electronic) | 978-1-0716-2176-9 |
| Publication status | Published - 19 May 2022 |
Publication series
| Name | Methods in Molecular Biology |
|---|---|
| Volume | 2466 |
| ISSN (Print) | 1064-3745 |
| ISSN (electronic) | 1940-6029 |
Abstract
This protocol describes the isolation of mitochondria by affinity chromatography using magnetic beads coated with Strep-Tactin in a timeframe of ca. 30 min. Compared to a classic differential and density gradient centrifugation this protocol enables a more rapid and efficient isolation of mitochondria even with small amounts of plant material. Transgenic plants with mitochondria that are decorated with a protein that is integrated into the outer mitochondrial membrane and fused to a green fluorescent protein (GFP) and a TwinStrep-tag facing the cytosol. This tag can bind to Strep-Tactin coated magnetic beads. Isolated mitochondria still bound to magnetic beads are uniquely suited for measuring oxygen consumption rates since this measurement needs mitochondria to be immobilized on the bottom of the measuring well. Furthermore, the isolated mitochondria can be used for downstream applications such as proteomics and metabolomics. This technique also allows for the isolation of mitochondria from specific cell types and tissues by altering the expression of the protein decorating the mitochondria.
Keywords
- Affinity chromatography, Arabidopsis, Lipidomics, Metabolomics, Mitochondria, Proteomics, Respiratory activity measurement
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Molecular Biology
- Biochemistry, Genetics and Molecular Biology(all)
- Genetics
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Methods in Molecular Biology. 2022. p. 121-133 (Methods in Molecular Biology; Vol. 2466).
Research output: Chapter in book/report/conference proceeding › Contribution to book/anthology › Research › peer review
}
TY - CHAP
T1 - Rapid Affinity Purification of Tagged Plant Mitochondria (Mito-AP)
AU - Niehaus, Markus
AU - Herde, Marco
N1 - Funding Information: We thank the Deutsche Forschungsgemeinschaft (HE 5949/3-1) for funding of the project.
PY - 2022/5/19
Y1 - 2022/5/19
N2 - This protocol describes the isolation of mitochondria by affinity chromatography using magnetic beads coated with Strep-Tactin in a timeframe of ca. 30 min. Compared to a classic differential and density gradient centrifugation this protocol enables a more rapid and efficient isolation of mitochondria even with small amounts of plant material. Transgenic plants with mitochondria that are decorated with a protein that is integrated into the outer mitochondrial membrane and fused to a green fluorescent protein (GFP) and a TwinStrep-tag facing the cytosol. This tag can bind to Strep-Tactin coated magnetic beads. Isolated mitochondria still bound to magnetic beads are uniquely suited for measuring oxygen consumption rates since this measurement needs mitochondria to be immobilized on the bottom of the measuring well. Furthermore, the isolated mitochondria can be used for downstream applications such as proteomics and metabolomics. This technique also allows for the isolation of mitochondria from specific cell types and tissues by altering the expression of the protein decorating the mitochondria.
AB - This protocol describes the isolation of mitochondria by affinity chromatography using magnetic beads coated with Strep-Tactin in a timeframe of ca. 30 min. Compared to a classic differential and density gradient centrifugation this protocol enables a more rapid and efficient isolation of mitochondria even with small amounts of plant material. Transgenic plants with mitochondria that are decorated with a protein that is integrated into the outer mitochondrial membrane and fused to a green fluorescent protein (GFP) and a TwinStrep-tag facing the cytosol. This tag can bind to Strep-Tactin coated magnetic beads. Isolated mitochondria still bound to magnetic beads are uniquely suited for measuring oxygen consumption rates since this measurement needs mitochondria to be immobilized on the bottom of the measuring well. Furthermore, the isolated mitochondria can be used for downstream applications such as proteomics and metabolomics. This technique also allows for the isolation of mitochondria from specific cell types and tissues by altering the expression of the protein decorating the mitochondria.
KW - Affinity chromatography
KW - Arabidopsis
KW - Lipidomics
KW - Metabolomics
KW - Mitochondria
KW - Proteomics
KW - Respiratory activity measurement
UR - http://www.scopus.com/inward/record.url?scp=85130296225&partnerID=8YFLogxK
U2 - 10.15488/17931
DO - 10.15488/17931
M3 - Contribution to book/anthology
C2 - 35585315
AN - SCOPUS:85130296225
SN - 978-1-0716-2175-2
T3 - Methods in Molecular Biology
SP - 121
EP - 133
BT - Methods in Molecular Biology
ER -