Generating Submitochondrial Vesicles from Saccharomyces cerevisiae Mitochondria to Isolate Mitochondrial Contact Sites

To begin, take 10 milligrams of freshly-isolated crude yeast mitochondria and re-suspend them in 1.6 milliliters of SM buffer at four degrees celsius by pipetting. Then, transfer the mitochondrial suspension to a pre-cooled 100-milliliter Erlenmeyer flask. Slowly, add 16 milliliters of the swelling buffer, using a 20-milliliter glass pipette while applying constant mild stirring on ice.

Incubate the samples under constant mild stirring for 30 minutes on ice. Then, slowly add five milliliters of 2.5-molar sucrose solution using a five-milliliter glass pipette, allowing the inner membrane to shrink. Incubate the samples under mild stirring for 15 minutes on ice.

To generate sub-mitochondrial membrane vesicles, transfer the mitochondrial suspension to a pre-cool rosette cell, and sonicate the suspension at 10%amplitude for 30 seconds while cooling the rosette cell in an ice bath. Rest the suspension for 30 seconds in an ice bath.