Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting

This protocol demonstrates the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis. Here the method applied to cultured human cells is described.

Transcript

Blue native polyacrylamide gel electrophoresis allows analysis of in-tact complexes of mitochondrial oxidative phosphorylation system. It is a convenient and inexpensive technique that can be used to overlay the assembly of entire complexes of mitochondrial oxidative phosphorylation system. To prepare the mitochondrial lysates, first use ice-cold PBS solution to gently wash the cells once.

Scrape the cells and pellet them at four degrees Celsius at 800 G for 10 minutes. Then, wash the cell pellet twice with the ice-cold PBS and centrifuge at four degrees Celsius at 800 G for 10 minutes. Next, add 200 microliters of 100x protease inhibitor to 20 milliliters of PBS.

Re-suspend the cells in the PBS protease inhibitor mixture to a final protein concentration of five milligrams per milliliter. To prepare 3.3 millimolar digitonin in PBS with protease inhibitor, dissolve four milligrams of digitonin in one milliliter of PBS at 100 degrees Celsius until no precipitate is visible. Cool on ice immediately and add 10 microliters of 100x protease inhibitor to one milliliter of the digitonin solution.

Next, add the digitonin protease inhibitor mixture to the cells at the final concentration of 1.65 millimolar. Mix well, and incubate on ice for five minutes. Then, add the PBS protease inhibitor mixture to the cells to the final volume of 1.5 milliliters.

Centrifuge at four degrees Celsius at 10, 000 G for 10 minutes. Remove the supernatant and re-suspend the mitochondrial pellet in mitochondrial buffer. Prepare one milliliter of fresh 10%loreal mountased in PBS inhibitor solution.

Add 10%loreal mountased to the mitochondria at the final concentration of 1%Incubate on ice for at least 15 minutes. Centrifuge at four degrees Celsius at 20, 000 G for 20 minutes. Collect the supernatant into a new tube and add the sample buffer.

To prepare the gradient gel for blue native page, first place the gradient maker on a stir plate and connect it with a flexible tubing to the peristaltic pump. Attach an infusion set with a needle to the tubing. Place a magnetic stirrer into the proximal chamber of the gradient maker and wash the tubing with distilled water at the maximum pump speed for 10 minutes.

Next, empty the tubing and the gradient maker. Use a pipette to remove any leftover distilled water in the channel between chambers of the gradient maker. Close the channel and the tubing with the valve.

Assemble two glass plates in the holder and place it on the stand. Using the hole on the bottom of the gel holder, place the needle connected to the tubing between the glass plates. To make an 8.3 by 7.3 centimeters gel, first prepare 6%and 15%gel solutions and keep them on ice.

Mix them gently to avoid making air bubbles. Then, load the proximal end of the gradient gel mixer tubing chamber with 2.6 milliliters of the 6%gel and the distal end with 2.1 milliliters of the 15%gel. Next, switch on the magnetic stirrer and open the tubing and the channel between chambers of the gradient maker.

Immediately switch on the peristaltic pump to five milliliters per minute. Fill the glass plates and remove the needle when there is no gel in the tubing. Gently overlay the gel with distilled water and keep the gel at the room temperature for at least one hour.

Wash the tubing immediately by filling the gradient chambers with distilled water and using the peristaltic pump at the maximum speed. Add six milliliters of the distilled water to three milliliters of the 3x gel buffer to make 1x gel buffer. With a filter paper, gently remove the distilled water from the surface of the gel.

Wash the surface of the gel with the 1x gel buffer, and gently remove the buffer with the filter paper. Prepare 4%stacking gel according to the manuscript. Mix gently to avoid making air bubbles.

Then, place the comb between the glass plates and pour the stacking gel under the comb. Immerse the comb fully. Let the stacking gel polymerize for at least 30 minutes.

Then, remove the comb and use a pipette to wash the wells with 1x gel buffer. To perform the blue native gel electrophoresis, first, add the blue cathode buffer to the gel cassette. Use a pipette to wash and fill the wells with the blue cathode buffer.

Then, load five to 30 micrograms of protein samples into the wells. Gently fill the gel cassette to the top with the blue cathode buffer, and the tank with the anode buffer. First, run the gel at a constant voltage of 40 volts for 15 minutes.

Then, increase the voltage to 80 volts. Run the gel until the dye reaches 2/3 of the gel length. Replace the blue cathode buffer with the cathode buffer and continue electrophoresis until the dye front has run off the gel.

Retrieve the glass plates and transfer the proteins to the PVDF membrane by semi-dry blotting. Use a constant voltage of 25 volts and a current limited to one ampere for 30 minutes. Assembly of the respiratory chain complexes in the human neuroblastoma cells were inhibited upon chloramphenicol treatment in comparison to the control cells without treatment.

In contrast, complexes encoded by the nuclear genes were up-regulated. Degradation of the oxidative phosphorylation complexes was observed upon multiple freeze-thaw cycles. The quality of the gel could affect the detection of the oxidative phosphorylation complexes.

Also, stripping of the membrane lowered the signal-to-noise ratio. When performing the procedure, it's important to remember to carry out simple preparation and gel electrophoresis under cold conditions to preserve in-tact OXPHOS complexes. Following blue native page, a second dimension SDS page can be applied to study the protein sub-units of respiratory chain complexes.

Blue native page allows researchers study the assembly of complexes and even super-complexes of oxidative phosphorylation system.

Journal (Biology)

Journal (Biology)

Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting

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