Department of Molecular Biology and Genetics, Science and Technology, Aarhus University, Slagelse, DenmarkDepartment of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark
Fernanda Salvato
Department of Biochemistry and Interdisciplinary Plant Group, University of Missouri-Columbia, Columbia, USAInstitute of Biology, State University of Campinas, Campinas, Brazil
Mingjie Chen
Department of Biochemistry and Interdisciplinary Plant Group, University of Missouri-Colum, Columbia, USA
R.S.P. Rao
Department of Biochemistry and Interdisciplinary Plant Group, University of Missouri-Columbia, Columbia, USA
Adelina Rogowska-Wrzesinska
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark
Ole Jensen
Department of Biochemistry and Interdisciplinary Plant Group, University of Southern Denmark, Odense M, Denmark
David Gang
Institute of Biological Chemistry, Washington State University, WA, USA
Jay Thelen
Department of Biochemistry and Interdisciplinary Plant Group, University of Missouri-Columbia, Columbia, USA
Ian Møller
Department of Molecular Biology and Genetics, Science and Technology, Aarhus University, Slagelse, Denmark
One way to study the function of plant mitochondria is to extract them from plant tissues in an uncontaminated, intact and functional form. The reductionist assumption is that the components present in such a preparation and the in vitro measurable functions or activities reliably reflect the in vivo properties of the organelle inside the plant cell. Here, we describe a method to isolate mitochondria from a relatively homogeneous plant tissue, the dormant potato tuber. The homogenization is done using a juice extractor, which is a relatively gentle homogenization procedure where the mitochondria are only exposed to strong shearing forces once. After removal of starch and large tissue pieces by filtration, differential centrifugation is used to remove residual starch as well as larger organelles. The crude mitochondria are then first purified by using a step Percoll gradient. The mitochondrial band from the step gradient is further purified by using a continuous Percoll gradient. The gradients remove contaminating amyloplasts and peroxisomes as well as ruptured mitochondria. The result is a highly purified, intact and functional mitochondrial preparation, which can be frozen and stored in liquid nitrogen in the presence of 5% (v/v) dimethylsulfoxide to preserve integrity and functionality for months.
