Product Description
Mitochondria are the main site of cellular respiration. The energy required for cellular activities is mainly supplied by oxidation occurring within mitochondria. The key to mitochondrial isolation is to maintain mitochondrial integrity and purity, which can be achieved by differential centrifugation: low‑speed centrifugation to remove nuclei and cell debris first, followed by high‑speed gradient centrifugation to isolate mitochondria.
Tissue Mitochondria Isolation Kit is designed for the rapid and convenient isolation of mitochondria from animal tissues. During isolation, cytosolic proteins depleted of mitochondria can also be obtained, which can be used to analyze the release of mitochondrial proteins (such as cytochrome C) into the cytoplasm. Most isolated mitochondria maintain intact inner and outer membranes and physiological functions (e.g., for mitochondrial membrane potential detection). The isolated proteins can be used for protein analysis including SDS‑PAGE, Western blotting, and two‑dimensional electrophoresis. This kit is suitable for extracting mitochondria from soft animal tissues (e.g., brain, liver) and hard tissues (e.g., heart, skeletal muscle).This kit is for research use only and is not intended for clinical diagnosis or other purposes.
Components
| O1509533 | Component | 50T | Storage |
| O1509533A | Mitochondria Lysis Buffer | 100 mL | -20℃. |
| O1509533B | Mitochondria Stock Buffer | 10 mL | -20℃. |
| O1509533C | Protein Stock Buffer (5×) | 10 mL | RT. |
| O1509533D | PMSF (100×) | 1.5 mL | -20℃. |
Materials Provided by User
1. Refrigerated centrifuge, homogenizer
2. PBS
Protocol (For Reference Only)
1. Washing: Take fresh tissue (frozen tissue is not recommended). Weigh 50–100 mg rapidly, wash once with pre-cooled PBS, and cut into tissue fragments of approximately 3 mm² on ice.
2. Homogenization and Lysis: Add 10 volumes of pre-cooled Mitochondria Lysis Buffer (if cytosolic protein is required, add PMSF in advance to a final concentration of 1×). Place in a Dounce homogenizer on ice and homogenize 10–20 times. The number of homogenization cycles varies with different tissues or homogenizers and should be optimized by the user.
3. Centrifugation: Centrifuge at 600×g, 4 ℃ for 5 min to remove nuclei, unbroken cells and large membrane debris.Note: For higher mitochondrial purity, centrifuge at 2000×g for 3 min, but the mitochondrial yield from the same amount of cells will be reduced.
4. Transfer the supernatant to a clean centrifuge tube. Centrifuge at 12000×g, 4 ℃ for 10 min. Note: For higher mitochondrial purity, centrifuge at 6000×g for 10 min, but the mitochondrial yield from the same amount of cells will be reduced.
5. Discard the supernatant, the pellet is mitochondria.If cytosolic protein depleted of mitochondria is desired, collect the supernatant in this step without disturbing the pellet. Then centrifuge the collected supernatant at 12000×g, 4 ℃ for 10 min. The resulting supernatant is cytosolic protein without mitochondria.
6. Storage
Discard the supernatant and resuspend the pellet in an appropriate buffer. For mitochondrial enzyme activity or functional analysis: resuspend the mitochondrial pellet in Mitochondria Stock Buffer. For mitochondrial protein analysis: store the obtained cytosolic protein in 1× Protein Stock Buffer by mixing cytosolic protein: Protein Stock Buffer (5×) at a ratio of 1: 4. For two-dimensional electrophoresis: use an appropriate storage solution.
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