Isolation of Membrane-enriched fractions from mouse cortical neurons
Odetta Antico, Erica Barini, Miratul M. K. Muqit
Abstract
Upon mitochondrial damage, activation of the PINK1 kinase and Parkin ubiquitin ligase induces ubiquitylation of multiple proteins at the mitochondria to stimulate their elimination by mitophagy. Protein ubiquitylation is a highly dynamic, reversible and complex post-translation modification (PTM) and it is frequently linked with phosphorylation. The major challenges, for biochemical and quantitative proteomic analysis of cellular proteins that are ubiquitylated and phosphorylated in response to mitochondrial damage in a PINK1-Parkin-dependent manner, involve the spatial configuration and stoichiometry of these post-translational modifications occurring on the mitochondria. Here, we describe an optimised protocol to isolate membrane-enriched fractions that provides high mitochondrial yield from primary cells, such as neuronal cultures. This protocol, in combination with other enrichment strategies, will facilitate proteomic and biochemical workflows for investigation of molecular events defined by PINK1/Parkin pathway.
Attachments
Steps
Membrane Isolation For Neurons-Mitochondrial depolarisation ◊TIMING 1-9h, day of experiment
Membrane Isolation For Neurons-Mitochondrial isolation ◊TIMING 1-1.5h
Gently aspirate media from wells and add 1mL of PBS+inhibitors in each well at 37Room temperature.
Scrape neuronal cells and collect in a 50mL labelled Falcon tube.
Centrifuge neuronal cells at 500x g,0h 0m 0s for 0h 3m 0s at 4°C.
Resuspend pellets in 2mL of HB buffer + inhibitors freshly added.
Homogenise cells using a stainless steel Dounce homogeniser tissue grinder with 40 stroke.
Pellet nuclei and the remaining intact cells from lysate at 2000rpm,0h 0m 0s for 0h 5m 0s at 4°C.
For total membrane isolation, transfer the PNS to an ultracentrifuge tube.
Spin at 200000x g,0h 0m 0s (40000rpm,0h 0m 0s) for 1h 0m 0s at 4°C.
Remove the supernatant, this represents the cytosolic fraction.
Pellet represents the membrane fractions. Freeze the pellet or resuspend the pellet according to the experiment.
For Western Blotting or proteomic experiments: transfer the pellet using a tip of a pipette to an Eppendorf tube and add 200µL-300µL of MitoBuffer. Sonicate the membrane pellet with a probe sonicator 20% amplitude for 0h 0m 5s or until the pellet is completely resuspended.
Proceed with protein quantification by using the Coomassie Protein Assay and sample preparation according to the experiment.
Membrane lysates can be stored at -80°C.
