The overall goal of native electrophoresis in combination with in-gel assays, immunoblotting, and native electroelution is to preserve and analyze the assembly of mitochondrial supercomplexes. This method can help answer how the protein complexes of the mitochondrial electron transport chain assemble into supercomplexes depending on many factors, including bioenergetic needs and physiological and pathological conditions. The main advantage of this technique is that if done properly, the physiological integrity and functionality of the mitochondrial supercomplexes is preserved throughout the lengthy isolation process.
This method can also be applied to study the dynamics of supercomplex assembly and to analyze protein complexes in the plasma membrane, as well as in other intracellular membranes. When preparing for in-gel assays, it is absolutely critical that all parts used for native electrophoresis are not contaminated by detergents, as they will contribute to a disintegration of the mitochondrial supercomplexes. Cut one or more lanes from a clear native gel and transfer these into a labeled container.
To perform the complex-1 assay, combine 10 milliliters of assay buffer with 25 milligrams of nitro blue tetrazolium and 100 microliters of 10 milligrams per milliliter NADH. Add this to the excised clear native gel lane or area of interest. Follow the development of blue bands after gentle agitation on a rocker for at least three minutes.
Then, fix the gel in 10 to 20 milliliters of acetic acid solution or wash in five millimolar Tris pH 7.4 to stop the reaction. Take photographs for documentation. To perform the complex-5 assay, obtain a gel, a lane, or an area of interest from a blue native or clear native gel.
Incubate the sample for two hours with gentle agitation in 10 to 20 milliliters of assay buffer, plus or minus five micrograms per milliliter of oligomycin at room temperature. After incubation, replace the buffer with 14 milliliters of fresh assay buffer. Then add, in order, 190 microliters of one molar magnesium sulfate, 27.28 milligrams of lead nitrate, 60 milligrams of ATP, and 75 microliters of oligomycin.
Incubate with gentle agitation and watch for a white precipitate as bands on the oligomycin-treated gels will give non-complex-5 dependent bands. Fix the gel in methanol based fixative because acidic solutions will dissolve the lead precipitate. Photograph the results and follow with protein transfer and immunoblotting, as described in the text protocol.
On the day of electroelution, place a frit in the bottom of each glass tube to be used. If necessary, place the glass tube in elution buffer and push the frit from inside to the bottom of the tube. Push the glass tube with the frit into the module of the Electro-Eluter.
Wet the grommet with elution buffer and slide the glass tube into place. Ensure that the tops of the glass tubes are even with the grommet. Close the empty grommets with stoppers.
Next, place a wet membrane cap at the bottom of the silicone adapter and fill the adapter with elution buffer. Slowly pipette the buffer and the adapter up and down to remove any air bubbles around the dialysis membrane. Slide the buffer-filled adapter to the bottom of the glass tube.
Then, remove all bubbles that appear on the frit inside the glass tube. Fill each glass tube with the elution buffer. Place the excised bands of the BN-PAGE into the glass tubes.
Cut large pieces into smaller pieces. Ensure that the fill height within the glass tube is around one centimeter. Then, place the entire module into the tank.
Add about 600 milliliters of cold elution buffer to the tank. Ensure that the silicone adapter caps are immersed in the buffer. Place a stir bar at the bottom of the tank before eluting the proteins for four hours at 350 volts in a cold room.
After the elution is completed, remove the Electro-Eluter module from the buffer tank and place it into a sink or bowl. If a stopper was used, remove it to drain the upper buffer chamber. Remove the buffer from each glass tube and discard it.
Make sure that the silicone adapter stays in place, and that the liquid below the frit is not disturbed or shaken. Then, carefully remove the silicone adapter, together with the membrane cap from the bottom of the glass tube. Pipette the contents of the silicone cap into a microtube.
With another 200 microliters of elution buffer, rinse the silicone cap and add to the microtube. Repeat this process for all glass tubes. Preserve the reusable membrane caps by placing them into elution buffer containing 0.5 milligrams per milliliter of sodium azide, and refrigerate them.
Supercomplexes from heart mitochondria are visualized here by in-gel assays. Lanes of the clear native gel are used for in-gel assays for complex-1 and complex-5. The specificity of the signal can be evaluated by using inhibitors, like oligomycin for the ATP-synthase.
In-gel assays provide information regarding the size of a protein complex, with complex-1 or complex-5 activity, but not about their composition. The blue native gel shows bands of protein complexes that can be excised for electroelution. Following electroelution, the eluate is used for further analysis, like in-gel assays, silver staining of the eluted proteins by native or denaturing electrophoresis, and Western blotting.
Once mastered, this technique can be done within three days if performed properly. These three days include isolating mitochondria, performing an overnight blue native maxi electrophoresis, and immunoblotting. After watching this video you should have a good understanding of how to isolate and identify mitochondrial supercomplexes using native electrophoresis, in-gel assays, and electroelution.
While attempting this procedure, it's important to remember that mitochondrial supercomplexes are fragile. Freezing and thawing of samples should be kept to a minimum. Native electrophoresis and electroelution should be done on ice or in the cold room.
The implications of these techniques extend beyond defining the physical composition of each supercomplex. Because the supercomplexes are captured in their native form, functional assays can be performed. After its development, this technique paved the way for researchers in the field of mitochondrial biology to re-evaluate the understanding of mitochondrial electron transport activity and its consequences for patients with mitochondrial diseases.
Following this procedure, other methods like HPLC, mass spectroscopy, electron microscopy or patch clamping can be performed to answer additional questions like the molecular composition, structure, or electrophysiological properties of the isolated protein complexes.
