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