This article describes a method for analyzing mitochondrial complexes of the respiratory chain using blue native gel electrophoresis. The technique allows for the preservation of the native structure and function of the complexes while enabling their separation based on size.
To analyze the five mitochondrial complexes of the respiratory chain with intact subunits, obtain mitochondrial complexes solubilized in mild, non-ionic detergent. Mix with buffer containing Coomassie Blue-G, an anionic dye.
Coomassie replaces the detergent and binds non-specifically to the complexes, imparting negative charge and staining them blue. Additionally, Coomassie prevents the complexes from aggregating and helps preserve their native structure and function.
In an electrophoresis tank, assemble a gel cassette comprising gradient resolving gel with decreasing pore size along the length, overlaid with large pore-size stacking gel. Fill the anode and cathode chambers with chilled anode and Coomassie-containing cathode buffer.
Load the wells with Coomassie bound-protein complex mixture. Initiate electrophoresis.
The negatively charged protein complexes migrate from the stacking gel into the gradient-resolving gel, toward the positively charged anode. While smaller complexes traverse rapidly through the pores, larger complexes are slowed down by restrictive pore sizes as they move down the resolving gel.
Coomassie dissociated from the complexes during the run is substituted by Coomassie in cathode buffer, preventing the complexes to shift from their respective positions in the gel. Post electrophoresis, visualize the gel. The mitochondrial complexes appear as blue bands resolved according to size.
Complex II with lowest molecular weight occupies the lowest position on the gel, followed by complex IV, III, and V. Complex I with highest molecular weight occupies the topmost position.
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 5 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 2/3rds 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.
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