Supercomplexes are supramolecular assemblies formed by respiratory chain complexes in the inner-mitochondrial membrane. Formation of these supercomplexes is believed to confer several structural and functional advantages, which include decreased production of reactive oxygen species, stabilization and more effective assembly of individual respiratory chain complexes, respiratory chain activity regulation, and prevention of protein aggregation in the protein-rich environment of the inner membrane. Changes in the assembly and composition of supercomplexes are dynamic, and are increasingly shown to underlie changes in mitochondrial function observed during physiological adaptation, as well as a variety of genetic and acquired diseases.
This video presents an hybrid clear/blue native PAGE protocol to resolve and analyze supercomplexes in a precise and time-effective manner. The main advantage of this hybrid clear/blue native PAGE method is that it optimally combines various aspects of traditional blue and clear native PAGE protocols, which allows to reduce exposure to detergents and anionic compounds to a minimum compared to published protocols. Optimal separation and analysis of supercomplexes is achieved on large gradient gels.
This procedure involves several delicate steps requiring manual skills and careful execution. Visual support to the written protocol provides important tips that facilitate the implementation of the technique. Demonstrating the procedure is Alexandria, a PhD student from my laboratory.
To begin, centrifuge the mitochondria isolated from the animal tissue in a 1.5-milliliter tube at 16, 000-times-G for 10 minutes at four degrees Celsius. Discard the supernatant and add the calculated volume of ice cold extraction buffer containing digitonin to the tube to resuspend the mitochondrial pellet. Place tubes on a mini tube rotator and incubate for 30 minutes at four degrees Celsius at a medium rotation speed.
Centrifuge samples at 20, 400-times-G for 45 minutes at four degrees Celsius to remove insolubilized fragments. Transfer the supernatant into a new tube on ice. If electrophoresis is not performed on the same day, store samples at minus 80 degrees Celsius.
Open the casting chamber. Place a 20 centimeters times 22 centimeters outer glass plate in the chamber. Position one set of 1.5-milliliter spacers using the alignment card to ensure they are seated firmly against the side and corners of the chamber.
Place an inner glass plate on top of the spacers to form the gel sandwich, and put a plastic separation sheet on top of the glass plate. Arrange three more gel sandwich assembly. Add more acrylic blocks or glass plates if needed to take up the remaining space in the chamber.
Place a strip of parafilm in the groove before seating the gasket firmly in the gasket notch. Place the ceiling plate on the chamber and tighten all six screws. Stand the casting chamber.
Place gradient former on a stir plate with a magnetic stirrer in the light mixing chamber. Connect the tubing of casting chamber to the gradient former, and secure the tubing in the cassette of the peristaltic pump. Make sure the stopcock of the gradient former is closed.
To cast four gels, prepare 60 milliliters of 4%and 60 milliliters of 12%gel solutions in two Erlenmeyer flasks, and swirl thoroughly to mix. Pour the 4%gel solution in the light mixing chamber, and the 12%in the heavy reservoir chamber of the gradient former. Set stir speed of the stir plate at 350 rpm.
Then open the stopcock and turn on the pump at 35 rpm. Once the solution level of the light fraction is lower than the heavy fraction, pause the pump and open the valve stem between light and heavy reservoirs. Let fractions volume equilibrate and restart the pump.
Make sure no bubbles enter the system and get trapped between glass plates. Once the gradient gel is completely poured, stop the pump and overlay one milliliter water on each gel sandwich to prevent drying of the gel. Let it polymerize for two hours.
Prepare 25 milliliter stacking gel in Erlenmeyer flask and swirl to mix thoroughly. Delicately invert the montage to remove water, and insert 15-well combs in each gel sandwich. Pour stacking gel and let it polymerize for two hours.
After that, insert gel and sandwich clamps and remove comb. With the short glass plate facing down, insert the gel sandwich in the cooling core. Repeat on the other side and place core in the electrophoresis tank.
Pour 300 milliliters of blue cathode buffer in inner chamber of the electrophoresis tank. With a pipette and loading tips, load 75 to 175 micrograms of protein per well. In cold room at four degrees Celsius, plug the electrodes into the power supply and turn it on to 150 volts, and run gel for one and a half hours, or until samples have all entered the gradient gel.
Load replicates of the sample in separate wells to enable parallel determination of in-gel activities and immunoblot analysis of OXPHOS complexes. Remove blue cathode buffer with pipette. Replace by 300 milliliters of Coomassie blue free cathode buffer, and run gel at 200 volts overnight in cold room.
Before the end of electrophoresis, prepare in-gel activity buffers according to the manuscript, and keep them in the dark at room temperature. Stop electrophoresis and retrieve gel. Cut the plastic bag so it can be opened like a book.
Cut lanes and transfer gel lanes into plastic bags. Use a heat sealer to seal two of the three sides. If negative control experiments are performed, add inhibitors to the assay buffers.
For three experimental samples, add 20 milliliters of in-gel activity buffer. Remove bubbles and seal the fourth side of plastic bag. Incubate gel lanes at 37 degrees Celsius in the dark and check every 15 minutes.
Incubation time varies depending on the amount of protein and complexes. After incubation, rinse gel lanes in water to stop reaction, and image on a white background for complex I, complex II, complex IV, or black background for complex V.For this example, a complex IV in-gel activity was performed to visualize supercomplexes isolated from fresh mouse liver mitochondria. The optimal amount of digitonin for this sample is four grams per gram, as it provides a good resolution of monomeric complex IV, and high molecular weight supercomplexes.
At a lower ratio, bands are not clear and resolve into a smear during electrophoresis, whereas the use of higher ratio of digitonin leads to disruption of high molecular weight supercomplexes. Liver mitochondria isolated from one mouse were treated with the optimal amount of digitonin to extract respiratory supercomplexes. The electrophoretic mobility of OXPHOS complexes is slightly reduced when proteins are separated using hybrid clear/blue native PAGE conditions, compared with standard native PAGE, due to reduced amount of Coomassie blue.
This mobility shift is greater for complex IV monomers, followed by complex V monomers, and complex I.The blue background is lower in the hybrid clear native/blue native PAGE compared to blue native PAGE. High background levels following blue native PAGE completely masks the in-gel activity staining for complex II, and enhances the background noise associated with the activity of complex IV dimers. For this protocol, titration of the samples of interest with various amounts of digitonin is critical in order to identify the conditions that allow optimal solubilization, while preserving enzyme activity and physiological protein interactions.
All step concerning the gel casting should be performed meticulously to the formation of an optimal gradient for proper sample separation. Extreme care should also be taken for the manipulation of these large and fragile gels. Always hold them with several fingers using the high percentage end of the gel.
With this hybrid clear/blue native PAGE method, samples are only exposed briefly to the anionic dye Coomassie blue at the beginning of electrophoresis without exposure to any other detergents. This allow to separate and resolve supercomplexes as effectively as with traditional blue native PAGE methods, while avoiding the negative effect of high Coomassie blue levels on in-gel activity assays and labile protein-protein interactions within supercomplexes. With this protocol, it is therefore possible to combine precise and rapid in-gel activity measurements with analytical techniques involving 2D electrophoresis, immunodetection, and/or proteomics referenced analysis of supercomplexes.
