TITLE: Blue Native PAGE of PINK1-TOM complex in mammalian cells
AUTHORS: Hina Ojha, Miratul M. K. Muqit

[DESCRIPTION]
Autosomal recessive mutations in PTEN-induced kinase 1 (PINK1) are associated with early-onset Parkinson's disease (PD) [1]. Upon inducing mitochondrial depolarization using uncouplers, the full-length human PINK1 stabilizes at mitochondria with the TOM (translocase of the outer mitochondrial membrane) complex, undergoing autophosphorylation and activation [2]. PINK1 plays a crucial role in phosphorylating ubiquitin and the E3 ubiquitin ligase, Parkin, initiating mitophagy for selectively removing damaged mitochondria [3]. To investigate PINK1 mutation impacts on PINK1-TOM complex formation, Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) was done on stable cell lines in PINK1 knockout (KO) HeLa cells expressing doxycycline-induced PINK1/mutants. BN-PAGE substitutes SDS with Coomassie G250, preserving proteins in their native conformation, and utilizes non-ionic detergents for solubilization, ensuring complex integrity during analysis [4, 5]. Studying the PINK1-TOM complex has proved vital for our understanding of PINK1 stabilization on mitochondria. Here, we share our method for resolving protein complexes by blue native PAGE for PINK1-TOM complexes.

[STEPS]
SECTION: Formation of mitochondrial enriched pellet
1. For making mitochondrial enriched fraction first harvest the cells after giving desired treatments (doxycycline for PINK1 induction and Antimycin/Oligomycin for mitochondrial damage). 
SECTION: Formation of mitochondrial enriched pellet
2. For collection keep plates with cells on ice covered with aluminium foil to provide even cool surface.

SECTION: Formation of mitochondrial enriched pellet
3. Wash the cells with PBS and collect the cells with cell scraper.
SECTION: Formation of mitochondrial enriched pellet
4. Collect the cells by centrifugation at 800 x g for 5 min at 4 °C.

SECTION: Formation of mitochondrial enriched pellet
5. Pellet down the cells at 800 x g for 5 min at 4 °C. For 150 mm plate cell pellet, add 300 µL of mitochondria fractionation buffer.

SECTION: Formation of mitochondrial enriched pellet
6. Disrupt cell membranes using a 25-gauge needle by passing through it for 25 times on ice.

SECTION: Formation of mitochondrial enriched pellet
7. Clarify lysates by centrifugation at 800 x g for 10 min at 4 °C.

SECTION: Formation of mitochondrial enriched pellet
8. Discard the cytoplasmic membrane/nucleus/debris pellet.
SECTION: Formation of mitochondrial enriched pellet
9. Isolate supernatant and centrifuge at 17000 x g for 20 min at 4 °C to collect mitochondrial enriched fraction.

SECTION: Formation of mitochondrial enriched pellet
10. Try to remove as much buffer as possible from the pellets as it may interfere with the BN-PAGE.
SECTION: Formation of mitochondrial enriched pellet
11. Snap-freeze the mitochondrial enriched pellet for Blue native PAGE.
SECTION: Mitochondrial enriched pellet Solubilization
12. Start with mitochondrial enriched pellets, snap-frozen and stored at -80 °C. A single freeze-thaw cycle has negligible effects on TOMs/PINK1. Whole cells can also be used but the signal is clearer in mitochondrial enriched fraction. 

 

SECTION: Mitochondrial enriched pellet Solubilization
13. Prepare Native-PAGE sample buffer per sample as described in buffer section.
SECTION: Mitochondrial enriched pellet Solubilization
14. Thaw mitochondrial pellets on ice and very gently resuspend them in BNPAGE buffer on ice with 1% digitonin. 

 

SECTION: Mitochondrial enriched pellet Solubilization
15. Store on ice and incubate for 30 min, gently pipetting up and down 10-15 times every 10 min. Digitonin is chosen after optimizing with different reagents.

SECTION: Mitochondrial enriched pellet Solubilization
16. Post-solubilization, centrifuge samples at 20000 x g for 30 min at 4 °C. Transfer the supernatant to a cold Eppendorf, noting a small red pellet of insoluble material. If desired, solubilize this material in 1X SDS-PAGE buffer for further analysis.

SECTION: Mitochondrial enriched pellet Solubilization
17. Quantify the lysate using BCA Protein Assay Kits.

 

SECTION: Mitochondrial enriched pellet Solubilization
18. Make samples of concentration around 1-2 ug/ul, typically loading 15-20 μg of solubilized mitochondrial pellets for one lane of a BN-PAGE gel. For diluting the lysates, make up the samples using 1x BN-PAGE lysis buffer with 1% digitonin. It is important to maintain the detergent percentage constant for proper Coomassie G250 and digitonin raito.
SECTION: Mitochondrial enriched pellet Solubilization
19. Prepare gels in 1X Native PAGE running buffer (20x stock, make up 1 L in Millipore water). Also, prepare light and dark inner chamber buffers. Keep all buffers at 4 °C.

SECTION: Mitochondrial enriched pellet Solubilization
20. Add Coomassie G250 additive to samples (at 1/4 the detergent concentration, i.e., 0.25% if using 1% digitonin). Pipette thoroughly to distribute Coomassie in the sample; the sample will turn a deep blue. 

 

SECTION: Mitochondrial enriched pellet Solubilization
21. Set up the gel apparatus in a cold room. Pour a small amount of Dark inner buffer into the inner chamber to check for leaks.
SECTION: Mitochondrial enriched pellet Solubilization
22. Remove any buffer from wells using an aspirator. 

 

SECTION: Mitochondrial enriched pellet Solubilization
23. Dry load samples onto the gel and use Native-Page ladder. 
SECTION: Mitochondrial enriched pellet Solubilization
24. After loading, fill the inner chamber with 200 mL Dark inner buffer and 1x running buffer in the outer chamber.

SECTION: Mitochondrial enriched pellet Solubilization
25. Run the gel at 7mA constant current and allow the sample to enter the gel until it runs 1/3rd of the total gel. Subsequently, empty both inner and outer buffers, replacing the inner buffer with LIGHT buffer and filling the outer chamber with 1x running buffer again.
SECTION: Mitochondrial enriched pellet Solubilization
26. Continue running the gel at 7mA for 2-3 hours until the full gel is stained.
SECTION: Transfer
27. Prepare 1x NuPAGE transfer buffer (20x stock) in a quantity sufficient for one transfer tank. Set up the transfer tank as usual.
SECTION: Transfer
28. For BN-PAGE, opt for PVDF membranes, as nitrocellulose binds Coomassie irreversibly. Activate the PVDF membrane in 100% methanol for 30 s before placing it over the gel in the transfer tank.
SECTION: Transfer
29. Upon completion, place the transfer tank in an ice bucket filled with ice. Run 30 s at ~60mA, limiting voltage to ~15 volts.
SECTION: Staining and Probing
30. Disassemble the transfer tank. If successful, most or all of the Coomassie staining on the gel will transfer to the PVDF membrane. 
SECTION: Staining and Probing
31. Destain Coomassie with 100% methanol 2-3 times for 5 min each. A slight Coomassie staining may persist (from the added samples; background staining will be removed).

SECTION: Staining and Probing
32. Restain the gel with Ponceau to visualize protein ladders. Mark ladders and scan the blot.
SECTION: Staining and Probing
33. Wash Ponceau with TBST (0.1% tween) until fully destained (3 times for 10 min each). It might take longer to destain completely. If needed, incubate in 5% milk in TBST to destain.

SECTION: Staining and Probing
34. Block in fresh 5% milk for 60 min (do not use BSA for blocking) and probe 60 min as for a standard Western blot.

SECTION: Staining and Probing
35. Wash membranes three times in TBST for 10 min each to remove residual primary antibody. The primary antibody may give different signals depending on how the antibody binding site is exposed in a complex. For visualization of PINK1 in PINK1-TOM complex, CST-PINK1 antibody works best.

SECTION: Staining and Probing
36. Utilize HRP-conjugated secondary antibodies. Prepare HRP-conjugated AB (1:5000 in milk), incubate at Room temperature covered for 60 min.

SECTION: Staining and Probing
37. Remove the secondary antibody and wash three times in TBST for 10 min each and once in TBS.

SECTION: Staining and Probing
38. Use ECL to visualize HRP-conjugated antibodies. Prepare the ECL reagent mix (1:1) just before scanning. Pour off TBS on one membrane and add 1 mL ECL mix; immediately scan, adjusting scanning times based on the primary antibody and protein abundance.