Isolation of primary cell heavy mitochondrial components from rat liver by differential centrifugation
Reagents and equipment
1. Mannitol buffer: 0.2 mol/L mannitol, 50 mmol/L sucrose solution, 10 mmol/L KCl, 1 mmol/L ethylenediaminetetraacetic acid, 10 mmol/L Hepes-NaOH, pH 7.4;
2. Glass rods;
3. Dounce homogenizer (20-30ml loosely formulated, Wheaton B type);
4. High torque bridge motor (semiconductor switch control);
5. High-speed centrifuge with fixed-angle rotor low-speed refrigerated centrifuge that can accommodate 40-50ml centrifuge tubes;
6. Potter-Elvehjem homogenizer, 20-40ml gap is 0.07mm;
7. Syringe (inner diameter of about 0.8mm) with metal trocar;
experimental method:
The given volume is suitable for about 10 g of liver. All operations after step 6 must be carried out at 0-4 °C.
1. Fasting a male rat overnight to deplete its glycogen;
2. The rats were sacrificed by cervical dislocation, and the liver was quickly removed and weighed;
3. Transfer the liver to a beaker placed on ice, carefully cut with scissors (the size of the liver fragments should not exceed 3 mm3), then suspend with mannitol buffer (about 4 ml buffer per gram of liver);
4. Pour half of the liquid and tissue debris into the Potter-Elvehjem homogenizer, and grind it up and down 5-6 times with a mortar (rotation speed is about 700r/min), and repeat the operation for the other half of the sample;
5. The homogenate was equally divided into 2 centrifuge tubes and centrifuged at 1000 g for 10 min;
6. After pouring the supernatant, place it on ice. Resuspend the pellet in 30 ml buffer, homogenize it with a mortar of a Potter-Elvehjem homogenizer for 2-3 times, and repeat step 5;
7. After pouring, combine the supernatant and centrifuge at 1000g for 10min;
8. Remove the supernatant with a metal trocar attached to a 20 ml syringe;
9. 4000g centrifugal supernatant for 15min;
10. Remove 4000 g of the supernatant from the centrifuge with a metal trocar and syringe. In order to remove the supernatant as much as possible, the needle is as close as possible to the concave surface;
11. Carefully suck out the loose pink material deposited on the dense brown precipitate, and wipe the inner wall of the tube with a piece of paper to remove the adhering liquid;
12. Add 10 ml of buffer to each tube and stir the pellet in a natural state with a glass rod.
13. Gently grind 3 times in a Dounce homogenizer with a mortar to complete resuspension;
14. Compensate the original volume with buffer and pour it into the new tube;
15. Repeat steps 9-14 three times;
16. Finally, the purified heavy mitochondria are suspended in a suitable medium (consistent with the subsequent analysis);
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