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.
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Acknowledgments
We thank the Deutsche Forschungsgemeinschaft (HE 5949/3-1) for funding of the project.
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Niehaus, M., Herde, M. (2022). Rapid Affinity Purification of Tagged Plant Mitochondria (Mito-AP). In: Ayyar, B.V., Arora, S. (eds) Affinity Chromatography. Methods in Molecular Biology, vol 2466. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2176-9_9
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DOI: https://doi.org/10.1007/978-1-0716-2176-9_9
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