Details
| Originalsprache | Englisch |
|---|---|
| Titel des Sammelwerks | Affinity Chromatography |
| Untertitel | Methods and Protocols |
| Seiten | 121-133 |
| Seitenumfang | 13 |
| ISBN (elektronisch) | 978-1-0716-2176-9 |
| Publikationsstatus | Veröffentlicht - 19 Mai 2022 |
Publikationsreihe
| Name | Methods in Molecular Biology |
|---|---|
| Band | 2466 |
| ISSN (Print) | 1064-3745 |
| ISSN (elektronisch) | 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.
ASJC Scopus Sachgebiete
- Biochemie, Genetik und Molekularbiologie (insg.)
- Molekularbiologie
- Biochemie, Genetik und Molekularbiologie (insg.)
- Genetik
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Affinity Chromatography: Methods and Protocols. 2022. S. 121-133 (Methods in Molecular Biology; Band 2466).
Publikation: Beitrag in Buch/Bericht/Sammelwerk/Konferenzband › Beitrag in Buch/Sammelwerk › Forschung › Peer-Review
}
TY - CHAP
T1 - Rapid Affinity Purification of Tagged Plant Mitochondria (Mito-AP)
AU - Niehaus, Markus
AU - Herde, Marco
N1 - Publisher Copyright: © 2022, Springer Science+Business Media, LLC, part of Springer Nature.
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 - Affinity Chromatography
ER -