Mitochondria Isolation Kit for Tissue

Preparation of mitochondria from soft tissues (liver or brain)

1. Use fresh tissue obtained within 1 h of sacrifice and keep it on ice. Do not freeze.

2. Weigh 50-100 mg tissue and wash with PBS.

3. Cut the tissue into the smallest possible pieces and suspend in 10 volumes of pre-cooled Mitochondria Isolation Reagent A containing 1 mM PMSF. For example, add 500 μL Reagent A to 50 mg tissue.

4. Homogenize on ice for approximately 10 strokes.

5. Centrifuge at 600 × g for 5 min at 4°C. For higher-purity mitochondria, 1,000 × g may be used, with a lower mitochondrial yield.

6. Carefully transfer the supernatant to a fresh tube and centrifuge at 11,000 × g for 10 min at 4°C. To reduce lysosome and peroxisome contamination, 3,500 × g may be used, with a lower mitochondrial yield.

7. Carefully remove the supernatant; the pellet contains mitochondria. To obtain mitochondria-free cytoplasmic protein, centrifuge the supernatant further at 12,000 × g for 10 min at 4°C and collect the supernatant for BCA or Bradford analysis.

Preparation of mitochondria from hard tissues (heart or skeletal muscle)

1. Use fresh tissue obtained within 1 h of sacrifice and keep it on ice. Do not freeze.

2. Weigh 50-100 mg tissue and wash with PBS.

3. Cut into small pieces, suspend in 10 volumes of pre-cooled PBS, incubate on ice for 3 min, centrifuge at 600 × g for 10-20 sec, and discard the supernatant.

4. Suspend in 8 volumes of pre-cooled Trypsin Buffer, incubate on ice for 20 min, centrifuge at 600 × g for 10-20 sec, and discard the supernatant.

5. Suspend in 2 volumes of the appropriate pre-cooled Mitochondria Isolation Reagent, centrifuge at 600 × g for 10-20 sec, and discard the supernatant. Use Reagent A for heart muscle, Reagent B for skeletal muscle, and Reagent A for other tissues.

6. Suspend in 8 volumes of the appropriate Mitochondria Isolation Reagent containing 1 mM PMSF and homogenize on ice for 20-30 strokes.

7. Centrifuge at 600 × g for 5 min at 4°C.

8. Carefully transfer the supernatant to a fresh tube and centrifuge at 11,000 × g for 10 min at 4°C.

9. Carefully remove the supernatant; the pellet contains mitochondria. Obtain mitochondria-free cytoplasmic protein as described for soft tissues.

Application of mitochondria

a. For applications requiring intact mitochondria, add 40 μL Mitochondria Storage Buffer per 100 mg tissue.

b. For mitochondrial protein characterization or functional assays, add 150-200 μL Mitochondria Lysis Buffer containing 1 mM PMSF per 50-100 mg tissue. The expected protein concentration is approximately 10-20 mg/mL. Before protein determination, centrifuge at 12,000 × g for 3-5 min at 4°C.