TITLE: CoxII degradation assay to assess mitophagy
AUTHORS: Thanh Ngoc Nguyen, nguyen.tha  

[DESCRIPTION]
This protocol details the procedure to assess mitophagy by analysing COXII degradation via Western blotting.

[STEPS]
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1. Seed the hela cells the day before the treatment day in 6 well plates.
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2. The next day, make sure the seeded cells are spreading out (not concentrated in the middle of the well because this can affect the results).
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3. Aspirate off the old media and treat each well with 2 mL of growth media containing 4 micromolar (µM) Antimycin A, 10 micromolar (µM) Oligomycin and 10 micromolar (µM) QVD for desired period.
 


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4. After the treatment, harvest the cells on ice by scraping.
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4.1. Pre-chill eppies and 1x PBS on ice.
 

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4.2. Aspirate the media thoroughly from the wells.
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4.3. Wash the wells with 1 mL of cold 1x PBS.
 


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4.4. Aspirate off the PBS and add another 1 mL of cold 1x PBS.
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4.5. Use a plastic cell scraper to scrape all the cells off the wells.
 

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4.6. Transfer the cells-containing PBS to eppies.
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4.7. Centrifuge the eppies at 3000 rpm for 2 min at 4 °C.
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4.8. Aspirate off PBS.
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4.9. Quickly centrifuge for 10 s to spin down the residual PBS.
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4.10. Aspirate off all the PBS.
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5. Lyse the cell pellets in lysis buffer and boil the samples at 99 °C with shaking for 7 min.
 


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6. Let the samples cool down and spin at max speed (Room temperature) for 1 min.
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7. Estimate the protein concentration by nanodrop
 

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8. Aliquot 25 µgof each sample into a new eppie and add 1x LDS to make up to 15 µL.
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9. Set up the gel tank with MOPs buffer.
 

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10. Wash each well with a glass syringe.
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11. Load markers and samples into the wells and run at 100V for 10 min and 190V for 55 min.
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12. Subject the gels to wet transfer onto PVDF membrane using cold NuPAGE transfer buffer containing 20% Methanol for 60 min at Room temperature.
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13. After transfer, incubate the PVDF membrane with PVDF destain buffer on a shaker at Room temperature for 2 min.
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14. Wash three times with PBS/Tween ( 5 min each wash).
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15. Block with blocking buffer for 15 min.
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16. Remove blocking buffer.
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17. Rinse twice with PBS/Tween and wash twice with PBS/Tween and once with 1x PBS (5 min for each wash).
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18. Cut the PVDF membrane and put appropriate parts into different antibodies.
 

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19. Incubate on a 4 °C shaker  .
 

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20. The next day, recycle the antibodies back to their tubes.
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21. Wash the blots three times with PBS/Tween (5 min for each wash).
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22. Incubate with appropriate HRP-conjugated secondary antibodies made up in blocking buffer for 60 min.
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23. Wash the blots twice with PBS/Tween, once with 1x PBS (5 min for each wash).
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24. Develop the blots with ECL prime.
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23.1. Wash the blot with PBS/Tween for 5 min (1/2).
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23.2. Wash the blot with PBS/Tween for 5 min (2/2).
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23.3. Wash the blot with 1x PBS for 5 min .
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21.1. Wash the blot with PBS/Tween for 5 min(1/3).
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21.3. Wash the blot with PBS/Tween for 5 min(3/3).
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21.2. Wash the blot with PBS/Tween for 5 min(2/3).
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17.1. Rinse the blocking buffer with PBS/Tween and wash with PBS/Tween for 5 min(1/2).
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17.2. Rinse the blocking buffer with PBS/Tween and wash with PBS/Tween for 5 min(2/2).
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17.3. Rinse the blocking buffer with 1x PBS and wash with 1x PBS for 5 min.
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14.1. Wash the PVDF membrane with PBS/Tween for 5 min (1/3).

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14.2. Wash the PVDF membrane with PBS/Tween for 5 min(2/3).
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14.3. Wash the PVDF membrane with PBS/Tween for 5 min(3/3).
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1.1. Each well contain 2 mL of growth media.
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1.3. Adjust the number of cells of other cell lines. So that the next day they are all in similar confluency with penta KO expressing BFP-Parkin and GFP-OPTN or -NDP52.
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1.2. Seed 350,000 cells for penta KO expressing BFP-Parkin and GFP-OPTN or -NDP52.
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25.