TITLE: Isolation of Membrane-enriched fractions from mouse cortical neurons
AUTHORS: Odetta Antico, Erica Barini, Miratul M. K. Muqit

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div></div>

[STEPS]
?. [Membrane Isolation For Neurons-Mitochondrial depolarisation ◊TIMING 1-9h, day of experiment]
To depolarize or uncouple mitochondrial membrane potential in neurons, cultures could be untilto   with a combination of   Antimycin A and   Oligomycin dissolved in DMSO at  .
?. [Membrane Isolation For Neurons-Mitochondrial isolation ◊TIMING 1-1.5h]
Gently aspirate media from wells and add  of PBS+inhibitors in each well at .
1 mL
37 Room temperature
Note: Do not use cold PBS, otherwise neuronal cells can detach from the dishes.
?. [Membrane Isolation For Neurons-Mitochondrial isolation ◊TIMING 1-1.5h]
Scrape neuronal cells and collect in a  labelled Falcon tube.
50 mL
?. [Membrane Isolation For Neurons-Mitochondrial isolation ◊TIMING 1-1.5h]
Centrifuge neuronal cells at  for  at .
Centrifuge: 500 34
4 °C
?. [Membrane Isolation For Neurons-Mitochondrial isolation ◊TIMING 1-1.5h]
Resuspend pellets in  of HB buffer + inhibitors freshly added.
2 mL
?. [Membrane Isolation For Neurons-Mitochondrial isolation ◊TIMING 1-1.5h]
Homogenise cells using a stainless steel Dounce homogeniser tissue grinder with 40 stroke.
Note: It is recommended to slowly press and twist the pestle on the sample. When the pestle is raised and turned, a strong vacuum force is generated creating shearing forces that will help to generate a fine homogenate. During this action, do not completely remove the pestle, this to avoid the generation of bubbles and to retain cellular organelles intact. Check cell lysis with Trypan blue (4ul lysate + 4ul trypan blue), 90% of cells should be disrupted.
?. [Membrane Isolation For Neurons-Mitochondrial isolation ◊TIMING 1-1.5h]
Pellet nuclei and the remaining intact cells from lysate at  for  at .
Centrifuge: 2000 33
4 °C
The subsequent supernatant is the post-nuclear supernatant (PNS).
?. [Membrane Isolation For Neurons-Mitochondrial isolation ◊TIMING 1-1.5h]
For total membrane isolation, transfer the PNS to an ultracentrifuge tube.
Note: Fill tube at least 4/5 with HB buffer to prevent tube collapse and equalize weight to
?. [Membrane Isolation For Neurons-Mitochondrial isolation ◊TIMING 1-1.5h]
Spin at  () for  at .
Centrifuge: 200000 34
Centrifuge: 40000 33
4 °C
?. [Membrane Isolation For Neurons-Mitochondrial isolation ◊TIMING 1-1.5h]
Remove the supernatant, this represents the cytosolic fraction.
?. [Membrane Isolation For Neurons-Mitochondrial isolation ◊TIMING 1-1.5h]
Pellet represents the membrane fractions. Freeze the pellet or resuspend the pellet according to the experiment.
?. [Membrane Isolation For Neurons-Mitochondrial isolation ◊TIMING 1-1.5h]
For Western Blotting or proteomic experiments: transfer the pellet using a tip of a pipette to an Eppendorf tube and add - of MitoBuffer. Sonicate the membrane pellet with a probe sonicator 20% amplitude for  or until the pellet is completely resuspended.
200 µl
300 µl
?. [Membrane Isolation For Neurons-Mitochondrial isolation ◊TIMING 1-1.5h]
Proceed with protein quantification by using the Coomassie Protein Assay and sample preparation according to the experiment.
?. [Membrane Isolation For Neurons-Mitochondrial isolation ◊TIMING 1-1.5h]
Membrane lysates can be stored at .
-80 °C