ExKine™ Mitochondrion Extraction/Isolation Kit for Tissue from Abbkine
Abbkine ExKine™ Mitochondrion Extraction Kit for Tissue enables rapid and crude isolation of of intact mitochondria from animal tissues from both soft and hard tissues using differential centrifugation. This crude mitochondrial preparation is often enough for most applications, such as study of mitochondrial respiration, mitochondria membrane potential, apoptosis, mtDNA and mtRNA, and mitochondrial protein profiling etc.
The Mitochondrion Isolation Kit for Tissue contains Lysis Buffer A (5×), Lysis Buffer B (5×), Storage Buffer.
Recommended procedures
A. For soft tissues (liver or brain)
1. Prepare a fresh tissue sample (obtained within one hour of sacrifice) and wash the sample twice with ice-cold PBS. Note: Do not freeze.
2. Cut 100 mg of tissue into very small pieces and wash the sample once with 1 mL icecold Lysis Buffer A.
3. Add fresh 1mL ice-cold Lysis Buffer A and transfer to a pre-cooling Dounce homogenizer.
4. Homogenize the sample on ice (usually 10-20 strokes).
5. Transfer the homogenate to a new tube and centrifuge the sample at 600g for 5 minutes. Note: For a more purified “heavy” mitochondrial fraction, this step can be changed to centrifuge at 1000 g for 5 minutes. The BSA (delipidated, final concentration 2mg/mL) can be added to the Lysis Buffer A to remove lipids, which may be present in the tissue.
6. Collect the supernatant in a new tube and centrifuge at 11,000 x g for 10 min at 4°C. Note: To obtain a more purified fraction of mitochondria, with >50% reduction of lysosomal and peroxisomal contaminants, this step can be changed to centrifuge at 3000 g for 15 minutes. The supernatant is cytosol fraction.
7. Remove the supernatant and resuspend the pellet in 1 mL ice-cold Lysis Buffer A. Repeat steps 5 and 6.
8. Suspend the pellet (purified mitochondria) in Storage Buffer (40 l per 100 mg tissue). Freeze and aliquot at -80°C until use. Note: It is expected the protein concentration of the sample should be approximately 10-25 mg/mL.
B. For hard tissues (skeletal or heart muscle)
Note: For heart muscle, use Lysis Buffer A and for skeletal muscle use Lysis Buffer B. For other tissues, use Lysis Buffer A first.
1. Prepare a fresh tissue sample (obtained within one hour of sacrifice) and wash the sample twice with ice-cold PBS. Note: Do not freeze.
2. Cut 100 mg of tissue into very small pieces. Centrifuge the sample at 600g for 30s, and then discard the supernatant.
3. Suspend the sample in 1mL 0.25 mg/mL trypsin in a new 2 mL Eppendorf tube.
4. Incubate on ice for 5-10min. Note: The incubation time varies in different tissues.
5. Spin down the tissue for a few seconds in the centrifuge and remove the supernatant.
6. Wash the sample once with 1 mL ice-cold the appropriate Lysis buffer and spin down the tissue for a few seconds in the centrifuge.
7. Remove the supernatant and add fresh 1 mL ice-cold the appropriate Lysis buffer.
8. Transfer the sample to a pre-cooling Dounce homogenizer and homogenize the sample on ice (usually 20-30 strokes).
9. Transfer the homogenate to a new tube and centrifuge the sample at 600g for 5 minutes. Note: For a more purified “heavy” mitochondrial fraction, this step can be changed to centrifuge at 1000 g for 5 minutes.
10. Collect the supernatant in a new tube and centrifuge at 11,000 x g for 10 min at 4°C. Note: To obtain a more purified fraction of mitochondria, with >50% reduction of lysosomal and peroxisomal contaminants, this step can be changed to centrifuge at 3000 g for 15 minutes. The supernatant is cytosol fraction.
11. Remove the supernatant and resuspend the pellet in 1 mL ice-cold appropriate Lysis Buffer. Repeat steps 9 and 10.
12. Suspend the pellet (purified mitochondria) in Storage Buffer (40 l per 100 mg tissue). Freeze and aliquot at -80°C until use.
Note: It is expected the protein concentration of the sample should be approximately 10-25 mg/mL.
Please click ExKine™ Mitochondrion Extraction/Isolation Kit (Tissue) Protocol for more detailed information.
Media Contact
Company Name: Abbkine Scientific
Contact Person: Media Relations
Email: Send Email
Phone: +86-27-59716789
Address:Bldg 1, Harbour of Technology Times, No.35, Optical Valley Ave
City: Wuhan
State: Hubei
Country: China
Website: https://www.abbkine.com/
