Sep 16, 2021
Isolation of Membrane-enriched fractions from mouse cortical neurons
- Odetta Antico1,
- Erica Barini1,
- Miratul M. K. Muqit1
- 1Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK
Protocol Citation: Odetta Antico, Erica Barini, Miratul M. K. Muqit 2021. Isolation of Membrane-enriched fractions from mouse cortical neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.bxjupknw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: August 20, 2021
Last Modified: May 31, 2024
Protocol Integer ID: 52564
Keywords: Neurons, Mitochondria, Membrane, PINK1, Parkin, Ubiquitin, ASAPCRN
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
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Materials
MATERIALS:
For Mitochondrial depolarisation and Membrane isolation:
1. Mitochondrial depolarisation: 10 micromolar (µM) Antimycin A from Streptomyces sp.Sigma – AldrichCatalog #A8674 ; 1 micromolar (µM) Oligomycin ASigma – AldrichCatalog #75351 in Dimethyl sulfoxide (DMSO)Sigma AldrichCatalog #D2650 .
2. HB (hypotonic Buffer) Buffer: 8.55% (w/v) sucrose: 42.25 g sucrose for 500 mL + 1:100 Imidazole (300 millimolar (mM) Stock). Filter the solution and store at 4 °C .
3. HB Buffer + inhibitors: HB + 200 millimolar (mM) Chloroacetamide, 1X complete protease inhibitors, 1 X phosphatase inhibitors cocktail 2 and 3 (add the day of the experiment).
4. MitoBuffer:
| A | B | |
| Sucrose | 270 mM | |
| HEPES | 20 mM | |
| EDTA | 3 mM | |
| Sodium orthovanadate | 1 mM | |
| 2-glycerophosphate | 10 mM | |
| Sodium fluoride | 50 mM | |
| Sodium pyrophosphate | 5 mM | |
| Chloroacetamide | 200 mM | |
| Pomplete protease inhibitors | 1X | |
| Phosphatase inhibitors cocktail 2 and 3 | 1 X |
5. PBS + inhibitors:
| A | B | |
| DPBS, no calcium, no magnesium (Gibco™ #14190094) | ||
| Sodium orthovanadate | 1 mM | |
| 2-glycerophosphate | 10 mM | |
| Sodium fluoride | 50 mM | |
| Sodium pyrophosphate | 5 mM | |
| PMSF | 10 mM | |
| Chloroacetamide | 200 mM | |
| Complete protease inhibitors | 1X | |
| Phosphatase inhibitors cocktail 2 and 3 | 1 X |
DPBS no calcium no magnesiumGibco - Thermo FischerCatalog #14190094
6. Table of reagents:
| A | B | C | |
| REAGENT | COMPANY | CAT. NUMBER | |
| D (+)-SACCHAROSE (SUCROSE) | VWR | 27480.36 | |
| IMIDAZOLE | Merck (Sigma-Aldrich) | 56750 | |
| HEPES | Formedium | HEPES10 | |
| SODIUM ORTHOVANADATE | Merck (Sigma-Aldrich) | S6508 | |
| SODIUM FLUORIDE | Merck (Sigma-Aldrich) | S7920 | |
| 2-GLYCEROPHOSPHATE DISODIUM SALT HYDRATE | Merck (Sigma-Aldrich) | G9422 | |
| PMSF | Merck (Sigma-Aldrich) | 93482 | |
| SODIUM PYROPHOSPHATE DECAHYDRATE | Merck (Sigma-Aldrich) | 221368 | |
| 2-CHLOROACETAMIDE | Merck (Sigma-Aldrich) | C0267 | |
| PHOSPHATASE INHIBITOR COCKTAIL 2 | Merck (Sigma-Aldrich) | P5726 | |
| PHOSPHATASE INHIBITOR COCKTAIL 3 | Merck (Sigma-Aldrich) | P0044 | |
| COMPLETE PROTEASE INHIBITORS | Merck (Roche) | 11873580001 |
D-( )-Sucrose AnalaR NORMAPUR® analytical reagentVWR ChemicalsCatalog #27480.360
ImidazoleSigma AldrichCatalog #56750
HEPESFormediumCatalog #HEPES10
Sodium OrthovanadateSigma AldrichCatalog #S6508-10G
Sodium pyrophosphate decahydrateSigma AldrichCatalog #221368
2-ChloroacetamideSigma – AldrichCatalog #C0267
Phosphatase Inhibitor Cocktail 2Sigma AldrichCatalog #P5726
cOmplete™ EDTA-free Protease Inhibitor CocktailRocheCatalog #11873580001
Sodium fluorideSigma – AldrichCatalog #S7920
β-Glycerophosphate disodium salt hydrateSigma – AldrichCatalog #G9422
Phenylmethanesulfonyl fluoride solutionSigma – AldrichCatalog #93482
Phosphatase Inhibitor Cocktail 3Sigma – AldrichCatalog #P0044
Cell lines:
1. Mouse cortical Neurons established from C57BL6J, PINK1 WT and KO, Parkin WT and KO mice as published in protocols.io (dx.doi.org/10.17504/protocols.io.bswanfae).
STOCK SOLUTION PREPARATION:
- Antimycin A: Prepare 50 millimolar (mM) of Antimycin A in DMSO; aliquot and store at -20 °C .
- Oligomycin: Prepare 10 millimolar (mM) of Oligomycin in DMSO; aliquot and store at -20 °C .
EQUIPMENT:
1. Eppendorf refrigerated centrifuge 5810R.
2. Beckman SW 55 Ti Swinging-Bucket Rotor 55000 rpm.
3. Optima L-90K Ultracentrifuge.
4. Cell culture incubator 5% CO2, 95% humidity HERAcell®CO2 incubator (150 L).
5. Microcentrifuges ventilated/refrigerated Micro Star 17 / 17RVWR international LtdCatalog #521-1647
6. Dounce Dura Grind® Tissue GrinderEMSCatalog #64791-07
7. Probe sonicator, Branson Digital Sonifier.
CONSUMABLES:
1. Cell culture multidishes, 6 well (Thermo Scientific™ #140675).Nunc™ Cell-Culture Treated Multidishes, 6 wellThermo FisherCatalog #140675
2. 50 mL Stripette™ Serological Pipets Polystyrene Individually Paper/Plastic Wrapped Sterile 25/BaCorningCatalog #4490
3. 25 mL Stripette™ Serological Pipets Polystyrene Individually Paper/Plastic Wrapped Sterile 25/BaCorningCatalog #4489
4. .10 mL Stripette™ Serological Pipets Polystyrene Individually Paper/Plastic Wrapped Sterile 50/BaCorningCatalog #4488
5. 5 mL Stripette™ Serological Pipets Polystyrene Individually Paper/Plastic Wrapped Sterile 50/BagCorningCatalog #4487
6. 15 mL conical centrifuge tubegreiner bio-oneCatalog #188271
7. 50 mL conical centrifuge tubegreiner bio-oneCatalog #227261
8. PIPETTE TIPS 100- 1000 µL BLUE SUITABLE FOR EPPENDORF STERILE 60 PIECES PER RACKgreiner bio-oneCatalog #686271 , PIPETTE TIP 10 - 100 µL SUITABLE FOR EPPENDORF 96 PIECES / ST RACKgreiner bio-oneCatalog #685261
9. 1.5ml Safe-lock tubesEppendorfCatalog #0030120086
10. Cell LiftersThermo FisherCatalog #08100240
11. 5 mL Open-Top Thinwall Ultra-Clear Tube 13 x 51mm - 50PkBeckman CoulterCatalog #344057
Membrane Isolation For Neurons-Mitochondrial depolarisation ◊TIMING 1-9h, day of experiment
Membrane Isolation For Neurons-Mitochondrial depolarisation ◊TIMING 1-9h, day of experiment
Note
To depolarize or uncouple mitochondrial membrane potential in neurons, cultures could be until
to 09:00:00 with a combination of 10 micromolar (µM) Antimycin A and 1 micromolar (µM) Oligomycin dissolved in DMSO at 37 °C .
Membrane Isolation For Neurons-Mitochondrial isolation ◊TIMING 1-1.5h
Membrane Isolation For Neurons-Mitochondrial isolation ◊TIMING 1-1.5h
1h 8m 5s
1h 8m 5s
Gently aspirate media from wells and add 1 mL of PBS+inhibitors in each well at Room temperature .
Note
Note: Do not use cold PBS, otherwise neuronal cells can detach from the dishes.
Scrape neuronal cells and collect in a 50 mL labelled Falcon tube.
Centrifuge neuronal cells at 500 x g for 00:03:00 at 4 °C .
3m
Resuspend pellets in 2 mL of HB buffer + inhibitors freshly added.
Homogenise cells using a stainless steel Dounce homogeniser tissue grinder with 40 stroke.
Note
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.
Pellet nuclei and the remaining intact cells from lysate at 2000 rpm for 00:05:00 at 4 °C .
Note
The subsequent supernatant is the post-nuclear supernatant (PNS).
5m
For total membrane isolation, transfer the PNS to an ultracentrifuge tube.
Note
Note: Fill tube at least 4/5 with HB buffer to prevent tube collapse and equalize weight to <0.01g.
Spin at 200000 x g (40000 rpm ) for 01:00:00 at 4 °C .
1h
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 200 µL -300 µL of MitoBuffer. Sonicate the membrane pellet with a probe sonicator 20% amplitude for 00:00:05 or until the pellet is completely resuspended.
5s
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 .