Mitochondria can be isolated from many plant tissues, but Arabidopsis cell suspension cultures have become a tissue of choice due to the relatively homogeneous tissue, the production of large amounts of plant material in a short time period, and the resources for proteomic analysis in Arabidopsis. All steps provided in the methodology should be performed at 4°C to minimize protein degradation.
Briefly, contamination-free 7-day-old cell suspension cultures are harvested by filtration through one layer of muslin cloth. The cells are homogenized in batches of 50 g FW by 3 successive 15-s bursts in a pre-cooled Warning blender in 150 mL HB comprising of 45 mM mannitol, 50 mM tetra-sodium pyrophosphate, 0.5% (w/v) PVPP 40, 2 mM EGTA, 0.5% (w/v) BSA, 20 mM cysteine, pH 8.0. To prevent excessive heating and frothing, 30-s rest intervals are applied between blending. The homogenate is filtered through three layers of Miracloth and one layer of muslin to remove cell debris. The filtrate is then centrifuged at 1500g to pellet residual cell debris. The supernatant is retained and centrifuged at 24,000g for 15 min, forming a crude organelle pellet.
The crude pellet is resuspended in a small volume of wash buffer (comprising 0.3 M mannitol, 10 mM TES-NaOH, pH 7.5, and 0.1% (w/v) BSA). The solution is then layered over a step Percoll gradient. The gradients consists of 5 mL 40% Percoll at the bottom, overlayed with 20 mL 25% Percoll, which is then overlayed with 5 mL 18% Percoll, all prepared in wash medium. Following centrifugation at 40,000g for 30 min, a mitochondrial-enriched band is formed at the 25% and 40% Percoll interface. Enzymatic marker assays reveal the presence of plastidic and peroxisomal enzymes but not cytosolic contamination . This band can be carefully aspirated, placed in new centrifuge tubes, and diluted in 5 times the volume with wash buffer for centrifugation at 24,000g for 15 min. The resulting pellet is overlayed onto a self-forming 35% Percoll gradient made in buffer containing 0.3 M sucrose, 10 mM TES-NaOH, pH 7.5, and 0.1% (w/v) BSA and centrifuged at 40,000g. The purified mitochondrial band is near the top of the gradient, while peroxisomal and plastidic contaminants are located lower in the gradient. The mitochondrial band can be carefully aspirated and transferred to new tubes, where it is washed in 5 times the volume in wash buffer without BSA by centrifugation at 24,000g for 15 min. The supernatant is discarded and the wash process is repeated. Typically, a yield of 10 mg mitochondrial protein is obtained from 100-g fresh weight of starting material. This is sufficient for a significant number of analyses of mitochondrial function and protein composition.
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