Blue Native Polyacrylamide Gel Electrophoresis: A Non-Denaturing Separation Technique for Analysis of Intact Mitochondrial Respiratory Chain Complexes

1. Preparation of buffers and solutions

NOTE: All the buffers and solutions used in the protocol are summarized in Table 1. The given volumes of the buffers are sufficient to prepare and run 10 samples. All the buffers can be stored at +4 °C for up to one year.

1. Prepare 20 mL of 3x gel buffer containing 1.5 M aminocaproic acid, 150 mM Bis-tris in distilled water (dH₂O). Adjust pH to 7.0.
2. Prepare 10 mL of 2 M aminocaproic acid in dH₂O.
3. Prepare 1 mL of the mitochondrial buffer by combining the following: 0.5 mL of 3x gel buffer, 0.5 mL of 2 M aminocaproic acid and 4 µL of 500 mM EDTA.
4. Prepare 1,000 mL of cathode buffer containing 15 mM of Bis-tris and 50 mM of tricine in dH₂O. Adjust pH to 7.0.
   1. Prepare 200 mL of blue cathode buffer by adding 0.04 g of Coomassie blue G-250 to 200 mL of cathode buffer.
5. Prepare 1,000 mL of anode buffer containing 50 mM Bis-tris in dH₂O. Adjust pH to 7.0.
6. Prepare 2.5 mL of sample buffer containing 750 mM aminocaproic acid and 5% Coomassie blue G-250 in dH₂O.

2. Preparation of mitochondrial lysates

1. Plate the cells the day before the collection. For HEK293 or 143B cells, use Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum, 1% L-glutamine, 100 mg/mL penicillin and 100 mg/mL streptomycin. Grow the cells in a cell culture incubator at +37 °C in a 5% CO₂ humidified atmosphere. Ensure that there are at least 500,000 cells for each sample on the day of collection.
2. Gently wash the cells once with ice-cold PBS. Avoid detaching the cells from the plate. Scrape the cells and pellet them at 800 x g for 10 min at +4 °C.
3. Wash the cell pellet twice with ice-cold PBS, and centrifuge as in step 2.2. Measure the protein concentration with the Bradford method using a commercial kit. Pellet the cells at 800 x g for 10 min at +4 °C.
   NOTE: After the cell collection, perform all steps at +4 °C and do not vortex.
4. Prepare 20 mL of PBS with protease inhibitor by adding 200 µL of 100x protease inhibitor to 20 mL of PBS. Keep on ice. Resuspend the cells in PBS with protease inhibitor to a final protein concentration of 5 mg/mL.
   1. To calculate the volume of PBS with protease inhibitor needed to resuspend the cells at 5 mg/mL, calculate the protein amount in each sample. For this calculation, use the protein concentration measured in step 2.3 and the volume of PBS used to resuspend the cells after washing in step 2.3.
5. Prepare 1 mL of 3.3 mM digitonin in PBS with protease inhibitor. Add 3.3 mM digitonin to a final concentration of 1.65 mM. Mix well and incubate on ice for 5 min.
   1. To prepare 1 mL of 3.3 mM digitonin in PBS with protease inhibitor, dissolve 4 mg of digitonin in 1 mL of PBS at 100 °C until no precipitate is visible and cool on ice immediately. Add 10 µL of 100x protease inhibitor to 1 mL of digitonin solution.
      NOTE: Use only a fresh solution of digitonin.
      CAUTION: Digitonin is toxic! Use a face mask, gloves and a lab coat.
6. Add PBS with proteinase inhibitor (prepared in step 2.4) to the final volume of 1.5 mL. Centrifuge at 10,000 x g for 10 min at +4 °C. Remove the supernatant. In this step, mitochondria are pelleted.
7. Resuspend the mitochondrial pellet in mitochondrial buffer. The volume of mitochondrial buffer is half of the volume of PBS in step 2.4.
8. Prepare fresh 10% lauryl maltoside in PBS containing protease inhibitor (prepared in step 2.4). 1 mL is sufficient for 10 samples. Add 10% lauryl maltoside to the final concentration of 1%. Incubate on ice for 15 min (this step can be longer up to a couple of hours).
9. Centrifuge at 20,000 x g for 20 min at +4 °C. Collect the supernatant into a new tube. Measure the protein concentration by the Bradford method using a kit. Add sample buffer, a volume that is half of the volume of lauryl maltoside used in step 2.8. Samples can be stored at -80 °C for up to 6 months.

3. Preparation of gradient gel for BN-PAGE

1. Pour the gradient gel for BN-PAGE at room temperature. Place the gradient maker on a stir plate and connect it with flexible tubing to the peristaltic pump. Attach an infusion set with the needle to the tubing. Place a magnetic stirrer into the proximal chamber of the gradient maker. Wash the tubing with dH₂O at maximum pump speed for 10 min.
2. Empty the tubing and the gradient maker. Use a pipet to remove any leftover dH₂O in the channel between chambers of the gradient maker. Close the channel and tubing with the valve.
3. Using casting frame and clamps assemble two glass plates in the holder, which has a hole on the bottom, and place it on the stand. Make sure that it is more or less on a straight surface with a water balance. Place the needle connected to the tubing between the glass plates.
4. Prepare 6% and 15% gel solutions (Table 2). Keep on ice. Add ammonium persulphate (APS) and tetramethylenediamine (TEMED) (they start polymerization) last. Mix gently to avoid making air bubbles.
5. Load the proximal end of the gradient-gel-mixer tubing chamber with 6% gel and the distal end with 15% gel. The total volume of the gel should be equal to the volume of the separating gel between the glass plates. Thus, use 2.6 mL of 6% gel and 2.1 mL of 15% gel to the gradient gel mixer for one 8.3 cm x 7.3 cm sized gel.
6. Switch on the magnetic stirrer, and open the tubing and channel between chambers of the gradient maker. Immediately switch on the peristaltic pump to 5 mL/min. Fill the glass plates, and do not allow bubbles to enter the gel. Remove the needle when there is no gel in the tubing.
7. Gently overlay the gel with dH₂O. Keep the gel at room temperature for at least 1 h for polymerization.
8. Immediately after pouring the gel, wash the tubing by filling the gradient chambers with dH₂O and using the peristaltic pump at maximal speed. When preparing two and more gels, always clean the system in between.
9. Prepare 1x gel buffer by mixing 3 mL of 3x gel buffer and add 6 mL of dH₂O. Remove dH₂O from the surface of the gel with filter paper gently. Wash the surface of the gel with 1x gel buffer. Remove 1x gel buffer with filter paper gently.
   1. Avoid fibers from the filter paper on the gel. To ensure this, cut the paper to small pieces with scissors, but do not tear the paper.
10. Place the comb between the glass plates, but do not immerse it fully to be able to pour the stacking gel under the comb. Prepare the 4% stacking gel (Table 2). Add APS and TEMED last as they start polymerization easily. Mix gently avoiding air bubbles.
11. Overlay the stacking gel, avoiding bubbles under the comb, and immerse the comb fully. Let the stacking gel polymerize for at least 30 min. Remove the comb and wash the wells with 1x gel buffer using a pipet.
    1. After polymerization, the gel can be stored at +4 °C for a couple of days. To store the gel, wrap the gel in paper soaked with dH₂O and plastic film to prevent drying of the gel.

4. Blue native gel electrophoresis

NOTE: To prevent degradation of OXPHOS complexes run electrophoresis at +4 °C. Use pre-chilled buffers.

1. Load the gel cassette with the blue cathode buffer until the bottom of the wells. Loading of the samples is easier when the cassette is not filled to the top. Wash the wells with the blue cathode buffer and then fill the wells with the blue cathode buffer using pipet.
2. Load the samples (5-30 µg of protein) into wells. Gently fill the gel cassette to the top with the blue cathode buffer and the tank with anode buffer.
3. Run the gel for 15 min at a constant voltage of 40 V. Then increase the voltage to 80 V (or use a constant current of 6 mA), do not exceed 10 mA. Run the gel until the dye reaches 2/3 of the gel length.
4. Replace the blue cathode buffer with cathode buffer and continue electrophoresis until the dye front has run out. In total, electrophoresis takes about 3 hours.
5. Retrieve the glass plates and transfer the proteins to the polyvinylidene fluoride (PVDF) membrane by semi-dry blotting.