TITLE: Isolated Mitochondria Characterization
AUTHORS: Megan Lee, Neal Bennett, Ken Nakamura

[DESCRIPTION]
This protocol is used to isolate mitochondria from cells. Isolated mitochondria can then be further analyzed, as described here, with a complex I enzyme activity assay, and/or blue native PAGE to quantify amounts of complex subunits.


[STEPS]
SECTION: Isolating Mitochondria
1. Make mitochondrial isolation buffers - starting buffer, mitochondrial resuspension buffer.

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1.1. 100 mM EGTA, pH 7.4:
3.8 g EGTA
70 mL water 
Adjusted pH KOH
Bring to 100 mL with water

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1.2. Starting Buffer (225 mM mannitol, 75 mM sucrose, 30 mM Tris-HCl, pH 7.4)
20.5 g mannitol
13 g sucrose 
400 mL water
15 mL Tris-HCl (1 M, pH 7.4)
Adjust pH to 7.4, and 
Bring to 500 mL with water


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1.3. Mitochondrial Resuspension Buffer (250 mM mannitol, 5 mM HEPES pH 7.4, 0.5 mM EGTA)
4.56 g mannitol 
80 mL water
1 mL 0.5M HEPES pH 7.4
Allow to cool
Adjust pH to 7.4
Bring to 100 mL with water
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2. Spin down 50 million cells.
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3. Resuspend cells in 5 mL PBS to wash.
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4. Spin down to create a cell pellet at 600 g for 5 minutes.
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5. Aspirate out the supernatant.
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6. Resuspend cells in 5 mL of PBS and spin down once more.
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7. Aspirate off the PBS.
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8. Resuspend the pellet in 1 mL of starting buffer and 100 mM EGTA (pH 7.4).
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9. Split the cell solution into 2 chilled 2 mL tubes each containing half the cell solution.
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10. Homogenize the cells using a tissue grinder for 200-250 strokes.
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11. Transfer the homogenate to new chilled centrifuge tubes.
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12. Centrifuge for 5 minutes at 600 g at 4˚C.
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13. Resuspend the pellet containing mitochondria in starting buffer.
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14. Centrifuge for 10 minutes at 7000 g at 4˚C.
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15. Discard supernatant.
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16. Resuspend the pellet in 1.6 mL starting buffer.
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17. Centrifuge for 10 minutes at 10000 g at 4˚C.
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18. Discard supernatant. Resuspend the pellet in 2 mL MRB buffer (mitochondria pellet).
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19. Snap freeze samples by putting them on dry ice and spraying the ice down with 70% ethanol.
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20. Store samples at -80˚C for further assays (i.e. enzyme activity assay, blue native PAGE).
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21. 
 
Make master mix with or without rotenone:
100 mM KPhos buffer pH 7.4 (make fresh monthly; store at 4°C)
200 mM KCN  (make fresh daily; 0.0130 g/1 mL 1M KPhos buffer pH 7.0) 
500 mM MgCl2
100 mg/mL BSA 
65 mM (21 mg/mL) decylubiquinone: Dissolve 10 mg of decylubiquinone in 0.476 mL ethanol to make a 65 mM (21 mg/mL) stock for long-term storage at -20°C.  On the day of the experiment, dilute 16 mL of 65 mM stock into 84 mL ethanol for a 100 mL working stock of ~10 mM
15 mM K2NADH  (make fresh daily; light sensitive; 0.0115 g/1 mL ddH2O) 
1 mM Antimycin A  (store at -20°C; 5.486 mg/10 mL 200 proof EtOH) 
0.5 mM Rotenone (store at -20°C; light sensitive; 5.916 mg/30 mL 200 proof EtOH)

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22. Dispense 100 µL of master mix into designated wells of a 96 well plate.
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23. Set spectrophometer (Spectramax M4 platereader) to read 340 nm wavelength for 5 min at constant 37°C.
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24. QUICKLY dispense 10 µg of crude mitochondria preps into each master mix well (minimize time here!).
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25. Record reading on spectrophotometer for 45 min.
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26. Prepare 20 µl sample buffer cocktail for every 50 µg crude mitochondria prep:
5 μl of NativePAGE sample buffer (4×), 8 μl of 5% digitonin, and 7 μl of water for a 8g/g digitonin to protein ratio.
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27. For dark blue cathode buffer, dissolve 0.044 g Coomassie Brilliant Blue G-250 in 220 ml of Native PAGE anode buffer, mix well.
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28. For light blue cathode buffer, add 20 ml of dark blue cathode buffer into 180ml of native PAGE anode buffer, mix well. The buffer can only be used once.
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29. Solubilize mitochondria by adding 20 µl sample buffer cocktail to 50 µg crude mitochondria prep and gently mix with a P20 pipette. Make sure to avoid formation of bubbles.
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30. Incubate solubilized mitochondria on ice for 20 min.
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31. Centrifuge at 20,000 × g for 10 min at 4°C and then collect 15 μl of the supernatant into new tubes.
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32. Add 2 μl of Coomassie G-250 sample additive to the above supernatant. The G-250 volume should be such that the final G-250 concentration is 1/4th the detergent concentration. This step should be done just before loading the samples into the gel.
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33. Set up the electrophoresis system (XCell SureLock Mini-Cell): 
Remove the white tape on the bottom of a NativePAGE 3-12% gradient gel and place it in the apparatus.
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34. Wash the wells of the gel with 1 ml of dark blue cathode buffer. Put 20 ml of dark blue cathode buffer in the inner chamber and check for leakage.
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35. Load 15 μl of sample into the gel by using Prot/Elec Tips. Fill the inner chamber with about 180 ml of dark blue cathode buffer, and then fill the outside chamber with about 600 ml of running buffer.
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36. Carefully add the dark blue cathode buffer without disturbing the samples in wells.
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37. Turn on the power supply and run the gel at 150 V for 30 min. Remove the dark blue buffer with a 10-ml size pipette or suction tube and fill the inner chamber with 200 ml of light blue buffer. Run the gel at 250 V for 60 min. If better separation of the bands is desired then the gel can be run for maximum of 150 min at 250 V. A longer run time than 150 min has minimal impact on the separation and resolution of bands.
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38. To transfer the BN-PAGE gel, first take the gel out of the cassette and wash it with water.
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39. Incubate the gel in transfer buffer for 15 min with gentle shaking, before transferring gel to PVDF membrane using an iBlot Gel Transfer Device.
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40. Block and stain PVDF membrane with total OXPHOS human western blot antibody cocktail at 1:500 concentration.
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41. Visualize blots with an Odyssey infrared imaging system.
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42. Analyze blots with Image Studio Lite Software from Li-cor Biosciences.