Isolate mitochondria from yeast

Buffer

Buffer 1: pH 9.3 100ml

Tris-HCl 100 mM 1.2114 g

DTT 10 mM (1M stock in water)

Buffer 2: pH 7.4 (KOH) 100 ml

Sorbitol 1.2 M 21.624 g

KH₂PO₄ 20 mM 0.2722 g

Buffer 3: pH 7.4 (KOH) 100 ml

Mannitol 600 mM 10.932 g

Hepes 20 mM 0.4766 g

BSA (fatty acid-free) 0.1%

PMSF 1 μM

Buffer 4: Buffer 3 without PMSF

Procedure

1. Culture 1000 ml yeast to early or middle growing stage: OD₆₀₀=0.5-1.5.

2. Centrifuge by 1500 g for 5 min at 4 ⁰C using 500 ml centrifuging tube.

3. Weigh 50 ml-centrifuging tube.

4. Transfer yeast pellet to the 50 ml centrifuging tube.

5. Weigh the weight of the yeast.

6. Resuspended in 2-4 volumes ice water, centrifuge by 1500 g at 4 ⁰C for 5 min.

7. Resuspended in 1 volume buffer 1, 30 ⁰C for 30 min while rocking.

8. Centrifuge, 1500 g for 5 min.

9. Wash once using buffer 2.

10. Resuspended in 3 volumes buffer 2, add Zymolyase-20T (20,000 U/g [ICN, CA]; 2 mg per g of cells) , 30 ⁰C for 1 hr, 50rpm, and check using water-breaking method every 15 min.

11. Centrifuge, 1500 x g for 5 min at 4 ⁰C.

12. Wash with buffer 2 one time or twice.

13. Resuspended in 2-volume buffer 3.

14. Homogenate (tight glass homogenizer, 20 times up and down, check the cell lysate by Trypan Blue).

15. Add buffer 3 and centrifuge at 1500 g for 5 min.

16. Repeat 13-15 for one time.

17. Save supernatant and centrifuge at 10000 g for 15 min at 4⁰C.

18. Wash one time using buffer 4.

19. Resuspended in buffer 4.

Isolating Mitochondria from Mouse (rat) Liver

  

Buffer

Isolating buffer: pH 7.4 100 ml

Sucrose 250 mM 8.5575 g

Hepes 10 mM 0.2383 g

EDTA 0.5 mM 0.0186 g

BSA (fatty acid free) 0.1% 0.1 g

Procedure

1． 把所有容器和器械4 ^oC 预冷，并用预冷的isolating buffer 洗过；

2． 取3－ 4个月大的小鼠，引颈处死；

3． 剖开腹腔，去胆囊，取肝脏（尽量使用肝尖部分，并除去非肝组织）；

4． 立即放入4 ^oC, isolating buffer 中，充分洗去血水；

5． 放入新鲜Buffer中剪碎；用 isolation buffer洗去血水

6． 4 ^oC ，电动匀浆机，上下三次；

7． 3000 rpm 离心10 min，取上清；应用 isolation buffer洗 2－ 3次

8． 10000 rpm 离心10 min，取沉淀即为线粒体；

9． 再用isolating buffer洗两次，均为 10000 rpm；

10． 最后用适量isolating buffer悬起 ;

11． Measuring the protein concentration;

12． Check oxygen consumption or PTP to see if the mitochondria are healthy.

NOTE ：Always on ice ！

1． You can pour off the supernatants and the loose upper part of the mitochondria pellet will come off as well. Most of the pellet, containing the healthy mitochondria is dense enough to remain behind. Using a posteur pipette to remove the last bit of the liquid. Wipe the inside of the upper part of the tube to remove any fatty material that remains. Any mixing of liquids with the mitochondria supernatants will cause them to uncouple quickly.

2． Use a Teflon rod to homogenize the remaining pellets. It should be completely smooth, a brown paste. Keep the tube on ice when stirring. Transfer the mitochondria to an Eppendorf tube using a yellow tip. Don't use glass to transfer because mitochondria stick to glass and you will lose most of your preparation. Keep the preparation on ice.

3． At this stage, the crude mitochondria preparation is done and can be suspend in the same buffer (without EGTA and protease inhibitor), and store on ice.

4． All above steps should be performed without delay; if there must be lapse of time between the preparation and the use, store the mitochondria as the pellet on ice.

Purification of Mitochondria by Sucrose Gradient Centrifuge

1． Preparation continues Sucrose Gradient from 1-2 M Sucrose buffered with 1 mM EDTA, 0.1% BSA (fatty acid free) and 10 mM Tris-HCl, pH 7.5, to purify mitochondria.

2． Using a gradient maker or   making four solutions of 1, 1.3, 1.6 and 2.0 M Sucrose buffered as above to format discontinuous gradient in the centrifuge tubes and let diffuse overnight at RT. Chilling the gradients to 4℃ before use.

3． Gently but thoroughly re-suspend the crude mitochoindria pellet in 0.8 M Buffered sucrose using the homogenizer with loose fitting pestle.

4． Overlay the homogenate on top of the gradients and centrifuge for 2 h at 80,000 g at 4℃ . The intact mitochondrial forms a band at about 1.9 g/ml while the occasionally broken mitochondria are found in denser or lighter brown bands.

5． Remove the intact mitochondria using a pipette

6． Dilute the gradient solution containing mitochondria with 2 Volume buffer contains 1 mM EDTA, 10 mM Tris-HCl, pH 7.4

7． Pellet the purified mitochondria by centrifugation at 20,000 g for 10 min at 4 ℃.

Purification of Mitochondria by Percoll Gradient Centrifuge

1. Prepare a linear Percoll gradient of 8-60% (V/V) buffered with 0.25 mM Sucrose, 0.2% BSA (fatty acid free) and 10 mM Tris-HCl, pH 7.5 using a gradient maker

2. Gently but thoroughly re-suspend the crude mitochoindria pellet in 0.8 M Buffered sucrose and lay on the top.

3. Centrifuge the gradients at 37,000 g for 20 min at 4 ℃.

4. Remove the mitochondria band and dilute it with 2 volume of the gradient buffer and pellet the mitochondria by centrifuge at 25,000 g for 10 min at 4 ℃ (or 6300g for sever times)

5. Wash with isotonic buffer to remove Percoll

6. Resuspend the purified mitochondria in MSB buffer containing 400 mM mannitol, 10 mM KH₂PO₄ ，50 mM Tris-HCl ，pH 7.2 and 5 mg/ml BSA (fatty acid free).

Subcellular Fractionation

Mitochondrial buffer (MB): pH 7.2 100 ml

Sucrose 210 mM 7.1883 g

Mannitol 70 mM 1.2753 g

EDTA 1 mM 0.0372 g

EGTA 1 mM 0.0380 g

MgCl₂ 1.5 mM 0.0305 g

Hepes (KOH) 10 mM 0.2383 g

量太少，不容易准确称量的，可先配好高浓度的贮存液再按比例添加。Make sure that you use Hepes (acid) and use KOH (0.5-1M) to adjust pH.

Protease inhibitor cocktail, some time PMSF only was added freshly

Procedure

1. Harvest cells (2 x 10⁶-1 x 10⁷cell).

2. Wash the cells with ice-cold PBS (Ca²⁺ free).

3. Resuspended in hypotonic mitochondrial buffer, and keep on ice for 30-60 min with frequent tapping. Wash once with the isolation buffer and resuspended in the isolation buffer with protease inhibitors were added. Using minimal resuspension buffer (approximately 5 times of the volume of the cell pellet).

4. Homogenized for 20-100 strokes with a Dounce homogenizer (typan blue staining, >30~50% positive cell). Wash the homogenizer once with minimal buffer.

5. Transfer to EP tube, which was put on ice before using.

6. 1000 x g, 5 –10 min, @ 4^oC to remove the nuclei and intact cells. if necessary, you may need to wash the pellet, combine the supernatant and spin down again to remove the unbroken cells (make sure that you remove the unbroken cell or nuclear by 1000 x g centrifugation before high speed centrifugation.

7. Collect and spin the supernatant at10, 000 g, 15 min, @ 4^oC (put centrifuge at 4 ^oC). Collect the pellet which contains Heavy Membrane including mitochondria.

8. Collect supernatant and spin at 100,000 x g, 1 h, @ 4^oC using bench-top ultracentrifuge and specific EP tube and the right rotor to obtain Light Membrane (ER fraction, pellet) and cytosolic fraction(supernatants).

9. Lyse the membrane with NP 40 lysis buffer (See the protocol of WB).

10. Adjust the protein concentration and add loading buffer for WB.

Note

1． Avoid using glassware, which might have excessive Ca²⁺. Make sure that you check the cell lysis with Trypan Blue to monitor the homogenization.

2． Keep everything on ice (0-4 ℃). Try to use minimal amount of buffer to avoid dilution of the cytosolic fractions