Sep 15, 2021
Reconstitution of Parkin ubiquitin ligase activity using mouse and human mitochondria
- Odetta Antico1,
- Alban Ordureau2,
- Michael Stevens1,
- J. Wade Harper3,
- 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;
- 2Sloan Kettering Institute;
- 3Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA
Protocol Citation: Odetta Antico, Alban Ordureau, Michael Stevens, J. Wade Harper, Miratul M. K. Muqit 2021. Reconstitution of Parkin ubiquitin ligase activity using mouse and human mitochondria. protocols.io https://dx.doi.org/10.17504/protocols.io.bxmypk7w
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 23, 2021
Last Modified: May 31, 2024
Protocol Integer ID: 52632
Keywords: MEFs, Mitochondria, PINK1, Parkin, Ubiquitin, ASAPCRN
Abstract
Analysis of Parkinson’s linked genes PINK1 and Parkin has uncovered a mechanism by which upon loss of mitochondrial membrane potential, Parkin E3 ubiquitin ligase activity is activated by PINK1 kinase activity, to trigger mitochondrial membrane protein ubiquitylation, leading to removal of damaged mitochondria (mitophagy). We and other groups have previously reported in vitro assays of Parkin E3 ligase activity using recombinant Parkin and PINK1 expressed in E. coli. This provided evidence of Parkin activation by PINK1 phosphorylation of Ser65 in both ubiquitin and UBL domain of Parkin. Herein, we report a reconstitution assay in which addition of recombinant Parkin to mitochondria isolated from cells after treatment by combination of Antimycin A and Oligomycin (to induce PINK1 activation on the outer mitochondrial membrane), enables robust ubiquitylation of multiple substrates at the mitochondria. This assay represents a powerful tool to study Parkin E3 ligase activity and the functional interplay between ubiquitylation and phosphorylation mediated by PINK1 and Parkin and their role in reshaping the endogenous mitochondrial proteome.
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Materials
For Mouse Embryonic fibroblast culture:
1. E13.5 mouse embryos (8–10 embryos, either sex; we used PINK1 wild-type and knockout mice) CRITICAL! All experiments must be conducted in accordance with the relevant institutional and governmental guidelines and regulations.
2. Digestion medium: 0.025% Trypsin-EDTAGibco - Thermo FisherCatalog #25300054 ; 0.125 mg/mL DNase IMerck Millipore SigmaCatalog #11284932001 in HBSS, calcium, magnesium, no phenol redGibco - Thermo FisherCatalog #14025050 .
3. Culturing medium:
| A | B | |
| DMEM (Gibco™ #11960-085) | ||
| Foetal Bovine Serum (FBS) heat inactivated (Gibco™ #10500064) | 20% | |
| Penicillin-Streptomycin (Gibco™ # 15140122) | 1% | |
| LGlutamine (Gibco™ #25030024) | 1% | |
| Non-essential Amino acid (Gibco™ #11140-035) | 1X | |
| Sodium pyruvate (Gibco™ #11360-039) | 1X |
DMEM, high glucose, no glutamineThermo FisherCatalog #11960085
Fetal Bovine Serum qualified heat inactivated BrazilGibco - Thermo FisherCatalog #10500064
Penicillin-StreptomycinGibco - Thermo FisherCatalog #15140122
L-Glutamine (200mM)Thermo Fisher ScientificCatalog #25030024
MEM Non-Essential Amino Acids Solution (100X)Thermo Fisher ScientificCatalog #11140035
Sodium Pyruvate (100 mM)Thermo FisherCatalog #11360039
4. DPBS no calcium no magnesiumGibco - Thermo FischerCatalog #14190094 .
5. Trypan Blue solutionSigma – AldrichCatalog #T8154
Cell lines:
1. Hela ATCC (Catalog# CCL-2)
Culturing medium:
| A | B | |
| DMEM (Gibco™ #11960-085) | ||
| Foetal Bovine Serum (FBS) (SigmaAldrich #F7524) | 10% | |
| Penicillin-Streptomycin (Gibco™ # 15140122) | 1% | |
| L-Glutamine (Gibco™ #25030024) | 1% |
DMEM, high glucose, no glutamineThermo FisherCatalog #11960085
Fetal Bovine SerumSigma AldrichCatalog #F7524
Penicillin-StreptomycinGibco - Thermo FisherCatalog #15140122
L-Glutamine (200mM)Thermo Fisher ScientificCatalog #25030024
For mitochondrial depolarisation and isolation:
1. Mitochondrial depolarisation: 10 micromolar (µM) Antimycin A (Sigma-Aldrich #A8674); 1 micromolar (µM) Oligomycin (Sigma-Aldrich #75351) in DMSO (Sigma-Aldrich #D2650).
Antimycin A from Streptomyces sp.Sigma – AldrichCatalog #A8674
Oligomycin ASigma – AldrichCatalog #75351
Dimethyl sulfoxide (DMSO)Sigma AldrichCatalog #D2650
2. Hypotonic Buffer:
| A | B | |
| HEPES (pH7.8) | 20 mM | |
| KCl | 5 mM | |
| MgCl2 | 1.5 mM | |
| DTT | 2 mM | |
| PMSF | 1 mM | |
| Phosphate inhibitors PhosSTOP | ||
| Protease inhibitor cocktail |
3. 2.5X MSH (Mannitol-Sucrose-HEPES) Buffer:
| Mannitol | 525 mM | |
| Sucrose | 175 mM | |
| HEPES (pH 7.8) | 20 mM | |
| EDTA | 5mM | |
| DTT | 2mM | |
| PMSF | 1mM |
4. 1X MSH Buffer:
| A | B | |
| Mannitol | 210 mM | |
| Sucrose | 70 mM | |
| HEPES (pH 7.8) | 20 mM | |
| EDTA | 2mM | |
| PMSF | 1mM | |
| Phosphate inhibitors PhosSTOP | ||
| Protease inhibitor cocktail |
5. Mito Ubi Buffer (MUB):
| A | B | |
| Tris-HCl pH 7.5 | 50 mM | |
| Sucrose | 70mM | |
| Sorbitol | 210 mM | |
| Sodium pyrophosphate | 5 mM | |
| Sodium Fluoride | 50 mM | |
| Sodium-2-glycerophosphate | 10mM |
6. DPBS no calcium no magnesiumGibco - Thermo FischerCatalog #14190094
7. Table of reagents
| A | B | C | |
| REAGENT | COMPANY | CAT. NUMBER | |
| D (+)-SACCHAROSE (SUCROSE) | VWR | 27480.36 | |
| D-SORBITOL | Merck (Sigma-Aldrich) | S1876 | |
| D-MANNITOL | Merck (Sigma-Aldrich) | M4125 | |
| EDTA DISODIUM SALT DIHYDRATE | Fisher BioReagents | BP120-500 | |
| TRIS (TROMETAMOL) | VWR | 103157P | |
| POTASSIUM CHLORIDE | VWR | 26764.298 | |
| MAGNESIUM CHLORIDE HEXAHYDRATE | Merck (Sigma-Aldrich) | 13152 | |
| DTT | Formedium | DTT010 | |
| HEPES | Formedium | HEPES10 | |
| 2-GLYCEROPHOSPHATE DISODIUM SALT HYDRATE | Merck (Sigma-Aldrich) | G9422 | |
| PMSF | Merck (Sigma-Aldrich) | 93482 | |
| SODIUM FLUORIDE | Merck (Sigma-Aldrich) | S7920 | |
| SODIUM PYROPHOSPHATE DECAHYDRATE | Merck (Sigma-Aldrich) | 221368 | |
| PHOSPHATASE INHIBITORS (phosSTOP) | Merck (Sigma-Aldrich) | 4906845001 | |
| COMPLETE PROTEASE INHIBITORS | Merck (Roche) | 11873580001 |
D-( )-Sucrose AnalaR NORMAPUR® analytical reagentVWR ChemicalsCatalog #27480.360
MannitolSigma AldrichCatalog #S1876
D-sorbitolSigma AldrichCatalog #M4125
Ethylenediaminetetraacetic Acid Di Na Salt Dihydr. (Crystalline Powd./Electrophor.) Fisher BioReagFisher ScientificCatalog #BP120-500
TRIS baseVWR ChemicalsCatalog #103157P
Potassium chloride 99.5-101.0% AnalaR NORMAPUR® Reag. Ph. Eur. analytical reagentVWR international LtdCatalog #26764.298
Magnesium chloride hexahydrateSigma – AldrichCatalog #13152
DTT 14-DITHIOTHREITOLFormediumCatalog #DTT010
HEPESFormediumCatalog #HEPES10
β-Glycerophosphate disodium salt hydrateSigma – AldrichCatalog #G9422
Phenylmethanesulfonyl fluoride solutionSigma – AldrichCatalog #93482
Sodium fluorideSigma – AldrichCatalog #S7920
Sodium pyrophosphate decahydrateSigma AldrichCatalog #221368
cOmplete ULTRA Tablets Mini EasyPack PhosStopSigma AldrichCatalog #4906845001
cOmplete™ EDTA-free Protease Inhibitor CocktailRocheCatalog #11873580001
For Ubiquitylation assay:
1. Tris-Base – 103157P VWR Prepare a 1 Molarity (M) Tris-HCl pH 7.5 stock in deionised water with the pH adjusted using 37.5 % HCl.
2. Magnesium chloride hexahydrateCatalog #M2670-500G
3. Adenosine Tri-Phosphat (ATP)AbcamCatalog #ab14730
1. TRIS(2-CARBOXYETHYL)PHOSPHINE HYDROCHLORIDEApollo ScientificCatalog #BIT0122
2. cDNA Clone - UBE1MRC PPU Reagents and ServicesCatalog #DU32888
3. UbE2L3 (MRC-PPU Reagents & Services, DU3772).
4. cDNA Clone - UbiquitinMRC PPU Reagents and ServicesCatalog #DU20027
5. cDNA Clone - parkinMRC PPU Reagents and ServicesCatalog #DU42598
For biochemistry
8. Coomassie Protein Assay ReagentThermo ScientificCatalog #1856209
9. 4X NuPAGE™ LDS Sample Buffer (4X)Invitrogen - Thermo FisherCatalog #NP0008
10. 2-mercaptoethanolSigma AldrichCatalog #M6250
11. PageRuler™ Prestained Protein Ladder, 10 to 180 kDaThermo Fisher ScientificCatalog #26616
12. Immobilon-P PVDF MembraneMerckCatalog #IPVH00010
13. Amersham™ Protran® Western blotting membranes nitrocelluloseMerckCatalog #GE10600041
14. NuPAGE™ 4 to 12% Bis-Tris 1.0 mm Mini Protein Gel 10-wellInvitrogen - Thermo FisherCatalog #NP0321BOX , NuPAGE™ 4 to 12% Bis-Tris 1.0 mm Midi Protein Gel 20-wellInvitrogen - Thermo FisherCatalog #WG1402BOX
15. NuPAGE™ MOPS SDS Running Buffer (20X)Invitrogen - Thermo FisherCatalog #NP000102 or NuPAGE™ MES SDS Running Buffer (20X)Thermo FisherCatalog #NP0002
16. 1 X Towbin transfer buffer:
| A | B | |
| Tris | 25 mM | |
| Glycine | 192 mM | |
| Methanol | 20% |
17. 1X Tris Buffered-Saline (TBS): 500 millimolar (mM) Tris, 150 millimolar (mM) Sodium chloride, 7.6 , at 25 °C .
18. 1X Tris-Buffered Saline, 0.1% Tween® 20 Detergent (TBST).
19. 5 % Non-Fat Milk in TBST.
20. 5% Bovine serum albumin (BSA) in Bovine Serum Albumin Fraction VSigma – AldrichCatalog #10735094001
21. Primary antibodies:
Phospho-Ubiquitin (Ser65) (E2J6T) Rabbit mAbCell Signaling TechnologyCatalog #62802
Purified anti-Ubiquitin AntibodyBioLegendCatalog #646302
Parkin Antibody (PRK8)Santa Cruz BiotechnologyCatalog #32282
CISD1/mitoNEET (D5M4C) Rabbit mAbCell Signaling TechnologyCatalog #83775
Anti-CPT1A antibody [8F6AE9] (ab128568)AbcamCatalog #ab128568
Cytochrome b5 Outer Mitochondrial Membrane AntibodyNovus BiologicalsCatalog #NBP1-88039
Hexokinase I (C35C4) Rabbit mAbCell Signaling TechnologyCatalog #2024
Recombinant Anti-Mitofusin 2 antibody [NIAR164] (ab124773)AbcamCatalog #ab124773
VDAC (D73D12) Rabbit mAbCell Signaling TechnologyCatalog #4661
22. Secondary Antibodies:
Goat anti-Rabbit IgG (H L) Secondary Antibody HRPInvitrogen - Thermo FisherCatalog #31460
Rabbit anti-Mouse IgG (H L) Secondary Antibody HRPInvitrogen - Thermo FisherCatalog #31450
23. ECL™ Western Blotting ReagentsMerckCatalog #RPN2106
24. SuperSignal™ West Dura Extended Duration SubstrateThermo FisherCatalog #34075
25. Hyperfilm™ ECL™MerckCatalog #28906837
STOCK SOLUTION PREPARATION:
- DNaseI: Dissolve 100 mg/mL (wt/vol) DNase in sterile double-distilled water; filter, aliquot and store at -20 °C . The solution is stable for 2–3 months.
- 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. Dumont #5 Forceps Biologie Inox (Fine Science Tool #11252-20).
2. Dumont #5XL Forceps Standard Inox (Fine Science Tool #11253-10).
3. Dumont #7 Fine Forceps Biologie Inox (Fine Science Tool #11274-20).
4. Student Vannas Spring Scissors Straight (Fine Science Tool #91500-09).
5. Scissors IrisFine Science ToolsCatalog #14058-09
6. Cell Counter-DeNovix CellDropTM.
7. 37 °C water bath.
8. Laminar flow cell culture hood.
9. Cell culture incubator 5% CO2, 95% humidity HERAcell®CO2 incubator (150 L).
10. Microcentrifuges ventilated/refrigerated Micro Star 17 / 17RVWR international LtdCatalog #521-1647
11. Dounce Dura Grind® Tissue GrinderEMSCatalog #64791-07
12. XCell4 SureLock™ Midi-CellThermo Fisher ScientificCatalog #WR0100
13. XCell SureLock™ Mini-CellThermo FisherCatalog #EI0001
14. Mini Trans-Blot Electrophoretic Transfer Cell #1703930Bio-rad LaboratoriesCatalog #1703930
15. Trans-Blot Cell With Plate Electrodes and Super Cooling Coil #1703939Bio-rad LaboratoriesCatalog #1703939
16. ChemiDoc MP Imaging System (BIORAD).
17. ECOMAX™ X-ray Processor.
18. Eppendorf ThermoMixer – 5382000031 Eppendorf.
CONSUMABLES
1. 10cm and 15cm tissue culture Petri Dishes (Nunc™ Cell Culture/Petri Dishes, 56.7cm2, Nunclon Delta treated, lid, ventThermo FisherCatalog #172931 and Nunc™ Cell Culture/Petri Dishes, 145 cm2, Nunclon Delta treated,lid, ventThermo FisherCatalog #168381 )
2. Falcon™ Cell StrainersFisher ScientificCatalog #10788201
3. Stericups 0.22µm, 250 mL and 500 mL (Stericup-GP Sterile Vacuum Filtration SystemFisher ScientificCatalog #SCGPU02RE , EMD Millipore™ Stericup™ Sterile Vacuum Filter UnitsFisher ScientificCatalog #SCGPU05RE )
4. 50 mL Stripette™ Serological Pipets Polystyrene Individually Paper/Plastic Wrapped Sterile 25/BaCorningCatalog #4490
5. 25 mL Stripette™ Serological Pipets Polystyrene Individually Paper/Plastic Wrapped Sterile 25/BaCorningCatalog #4489
6. 10 mL Stripette™ Serological Pipets Polystyrene Individually Paper/Plastic Wrapped Sterile 50/BaCorningCatalog #4488
7. 5 mL Stripette™ Serological Pipets Polystyrene Individually Paper/Plastic Wrapped Sterile 50/BagCorningCatalog #4487
8. 15 mL conical centrifuge tubegreiner bio-oneCatalog #188271
9. 50 mL conical centrifuge tubegreiner bio-oneCatalog #227261
10. Standard 1mL and 200μL Pipette tips (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 )
11. Syringe filter (0.22μm. Sartorius, Item # ST16541-Q).
12. Syringes (50mL) (Terumo™# 8SS50L1).
13. 1.5ml Safe-lock tubesEppendorfCatalog #0030120086
14. Cell LiftersThermo FisherCatalog #08100240
PROCEDURE TO ISOLATE AND CULTURE MOUSE EMBRYONIC FIBROBLASTS-Dissection of E13.5 mouse embryos ◊TIMING 20 min
PROCEDURE TO ISOLATE AND CULTURE MOUSE EMBRYONIC FIBROBLASTS-Dissection of E13.5 mouse embryos ◊TIMING 20 min
20m
20m
Use sterilized instruments by autoclave or washing them with 70% (vol/vol) ethanol. Dry thoroughly if ethanol is used.
Soak dissection tools in 70% ethanol between embryos to prevent contamination.
In the hood. Prepare 10 cm dishes with cold PBS. Separate embryos from uterus and placenta. Place each embryo into a single dish with cold PBS.
Number dishes and Eppendorf tubes for tissue collection for genotyping.
Euthanize the embryos by decapitation and separate the head from the body.
Wash the bodies twice with PBS to minimise contamination and collect a small piece of tail for genotyping.
Place the body on a dish with PBS and remove the red spot (bowel) with forceps.
Place the body on a clean dish and mince the tissue with a spring scissors (or with a sterile scalpel blades).
Cell dissociation and plating
Cell dissociation and plating
30m
30m
Prepare digestion medium by adding 125 µL of DNase I (stock solution 10 mg/mL) to 10 mL of Trypsin 0.025% (1:1 Trypsin 0.05%-HBSS).
Add 5 mL of digestion medium to the tissue and transfer in a 15 mL falcon tube.
Incubate at 37 °C in a water bath for 00:15:00 .
15m
Pipette to mechanically dissociate the tissue, gentle and sequential pipetting (using 10 mL , 5 mL and 1 mL pipettes) until cells are completely suspended.
Note
Note: The number of trituration is approximative, it may vary depending on the size of the unbroken tissues.
Inactivate trypsin digestion by 5 mL of culturing medium.
Centrifuge at 1200 rpm for 00:05:00 .
5m
Remove media and resuspend in 5 mL culturing media.
Filter the cells through a 70 μm filter.
Note
Note: The cell suspension should be filtered in order to exclude any undigested tissue pieces or aggregates from the newly prepared cell suspension.
Take a 15 µL aliquot, add 1:1 ratio Trypan Blu and determine the density of cells and cell viability to the cell counter.
Plate the 3.0 × 106 cells/well plates out on 10 cm dishes, containing 10 mL of pre-warmed culturing media.
Change media every 5 days, grow to 90% confluence and split (minimum 25% confluence to keep cells within the range to promote growth). The growth rate progressively declines when transformation occurs and the cells become immortal, at approximately after 18 passages (it can be variable).
Note
Note: In this assay we use primary MEFs between 8-10 passages.
MITOCHONDRIAL ISOLATION FOR MEFs-Mitochondrial depolarisation ◊TIMING 4h, day of experiment
MITOCHONDRIAL ISOLATION FOR MEFs-Mitochondrial depolarisation ◊TIMING 4h, day of experiment
4h
4h
To depolarize or uncouple mitochondrial membrane potential in MEFs, treat the cultures for 04:00:00 with a combination of 10 micromolar (µM) Antimycin A and 1 micromolar (µM) Oligomycin dissolved in DMSO at 37 °C .
Note
Note: Before the experiment, MEFs were plated in 15 cm dishes and stimulated at 80-90% confluence.
4h
MITOCHONDRIAL ISOLATION FOR MEFs-Mitochondrial isolation ◊TIMING 1-1.5h
MITOCHONDRIAL ISOLATION FOR MEFs-Mitochondrial isolation ◊TIMING 1-1.5h
Gently aspirate the medium from wells.
Wash twice by adding 5 mL of warmed DPBS (room temperature) containing protease inhibitors and phosphatase inhibitors.
Place the 15 cm dish On ice and add 1 mL of Hypotonic Buffer. Carefully scrape the cells and collect the cells in a 15 mL microcentrifuge tube. Add 2 mL of Hypotonic Buffer in each tube (for a total of 3 mL ).
Stand On ice for 00:15:00 in the cold room.
15m
Homogenise cells using a stainless steel Dounce homogeniser with 45 strokes.
Note
Note: Check cell disruption with light microscope, 80-90 % cells should be disrupted.
Add to the disrupted cells 2.5X MSH buffer and mix.
Note
Note: Mixing 2.5X MSH volume to the initial volume of hypotonic buffer will give 1X MSH.
Centrifuge the homogenate at 700 x g in a refrigerated centrifuge for 00:10:00 , to remove cell debris and nuclei.
10m
Transfer supernatant into a new 15 mL tube and centrifuge at 700 x g x 00:10:00 at 4 °C to remove residual nuclei and cell debris.
10m
Centrifuge at 9000 x g x 00:10:00 at 4 °C to pellet mitochondria.
10m
Resuspend mitochondria in 1 mL of 1X MSH buffer and centrifuge at 9000 x g x 00:10:00 at 4 °C to pellet mitochondria. Repeat twice to remove any cytosolic proteins.
10m
Centrifuge at 9000 x g x 00:10:00 at 4 °C to pellet mitochondria and resuspend in 150 µL of MUB Buffer.
10m
Protein quantification: take a small aliquot of mitochondria (10 µL), add 1% Triton, vortex and estimate protein concentration by using the Coomassie Protein Assay.
Note
Usually from a 15 cm dish of MEFs at 90% confluence it is possible to isolate 200µg of crude mitochondria.
MITOCHONDRIAL ISOLATION FOR HeLa-Mitochondrial depolarisation ◊TIMING 2h, day of experiment
MITOCHONDRIAL ISOLATION FOR HeLa-Mitochondrial depolarisation ◊TIMING 2h, day of experiment
2h
2h
To depolarize or uncouple mitochondrial membrane potential in MEFs, treat the cultures for 02:00:00 with a combination of 10 micromolar (µM) Antimycin A and 1 micromolar (µM) Oligomycin dissolved in DMSO at 37 °C .
Note
Note: Before the experiment Hela were plated in 15 cm dishes and stimulated to 80- 90% confluence.
2h
MITOCHONDRIAL ISOLATION FOR HeLa-Mitochondrial isolation ◊TIMING 1-1.5h
MITOCHONDRIAL ISOLATION FOR HeLa-Mitochondrial isolation ◊TIMING 1-1.5h
2h
2h
Gently aspirate the medium from wells.
Wash twice by adding 5 mL of warmed DPBS (Room temperature ) containing protease inhibitors and phosphatase inhibitors.
Place the 15 cm On ice and add 1 mL of Hypotonic Buffer. Carefully scrape the cells and collect the cells in a 15 mL microcentrifuge tube. Add 2 mL of Hypotonic Buffer in each tube (for a total of 3 mL ).
Stand On ice for 00:15:00 in the cold room.
15m
Homogenise cells using a stainless steel Dounce homogeniser with 25 strokes.
Note
Note: Check cell disruption with light microscope, 80-90 % cell should be disrupted.
Add to the disrupted cells 2.5X MSH buffer and mix.
Note
Note: Mixing 2.5X MSH volume to the initial volume of hypotonic buffer will give 1X MSH.
Centrifuge the homogenate at 700 x g in a refrigerated centrifuge for 00:10:00 , to remove cell debris and nuclei.
10m
Transfer supernatant into a new 15 mL tube and centrifuge at 700 x g x 00:10:00 at 4 °C to remove residual nuclei and cell debris.
10m
Centrifuge at 9000 x g x 00:10:00 at 4 °C to pellet mitochondria.
10m
Resuspend mitochondria in 1 mL 1X MSH buffer and centrifuge at9000 x g x 00:10:00 at 4 °C to pellet mitochondria. Repeat twice to remove any cytosolic proteins.
10m
Centrifuge at 9000 x g x 00:10:00 at 4 °C to pellet mitochondria and resuspend in 150 µL -250 µL of MUB Buffer.
10m
Protein quantification: take a small aliquot of mitochondria (10 µL ), add 1% Triton, vortex and estimate protein concentration by using the Coomassie Protein Assay.
Note
Usually from one 15 cm dish of HeLa at 80-90% confluence it is possible to isolate 600-800µg of crude mitochondria.
MITOCHONDRIAL ISOLATION FOR HeLa-UBIQUITYLATION ASSAY ◊TIMING 2.5h-3h
MITOCHONDRIAL ISOLATION FOR HeLa-UBIQUITYLATION ASSAY ◊TIMING 2.5h-3h
Resuspend isolated mitochondria in MUB buffer at concentration ~1 mg/mL , in order to use a volume of mitochondria < 10% of reaction volume.
Use 5 µg of mitochondria for a total volume reaction of 50 µL .
Defrost proteins 0 °C and prepare a master mix considering that for one single reaction it is required 1 micromolar (µM) Parkin, 0.1 micromolar (µM) His-UbE1, 1 micromolar (µM) UBE2L3 and 30 micromolar (µM) Ubiquitin (as negative control prepare a master mixer without Parkin).
Prepare reaction buffer with 50 millimolar (mM) Tris 7.5 , 5 millimolar (mM) MgCl2 and 0.5 millimolar (mM) TCEP.
Combine reaction buffer, master mix of proteins and mitochondria.
Aliquot in order to distribute 50 µL in 1.5 mL Eppendorf tube and start the ubiquitylation reaction by adding 2 millimolar (mM) ATP in each Eppendorf tube.
Place the eppendorf tubes in a thermomixer and incubate the reaction at 30 °C , shaking for 02:00:00 (MEFs) or 01:30:00 (HeLa) at 1000 rpm .
3h 30m
Stop the reaction by the addition of 4X LDS loading buffer containing 10% of 2- mercaptoethanol.
MITOCHONDRIAL ISOLATION FOR HeLa-Immunoblotting of ◊TIMING 5h-2d
MITOCHONDRIAL ISOLATION FOR HeLa-Immunoblotting of ◊TIMING 5h-2d
4h 53m
4h 53m
Boil the samples for 00:03:00 at97 °C .
3m
Analyse samples by running 20 µL of reaction on Nu-page Bis-Tris 4-12% gels for a better resolution of ubiquitin chains, at 120 V for ~2h. Use MES SDS Running Buffer or MOPS SDS Running Buffer according to the size of protein to be analysed.
Note
For phospho-parkin and Parkin blots, it is recommended to dilute the final reaction 1 in 25 with 1X LDS containing 2.5 % 2- mercaptoethanol.
Transfer gel on PVDF membrane for phospho-ubiquitin and ubiquitin signal and nitrocellulose membrane for phospho-Parkin and Parkin signal. Transfer in Towbin buffer at 80 V for 01:30:00 On ice or in cold room (for HK1 it is recommended to transfer at 90 V for 1.5h).
Note
Note: Prepare only 1 membrane per transfer tank –avoid multiple membranes for transfer in same tank as this reduces ubiquitin transfer.
1h 30m
Incubate membrane with blocking buffer 5% milk in 0.1% TBS-Tween for 01:00:00 at Room temperature
1h
Remove blocking buffer, if primary antibodies are in 5% BSA, rinse twice with 0.1% TBS-Tween to remove any traces of milk, add primary antibodies and incubate Overnight at 4 °C .
Note
Note: Prepare phospho-Ubiquitin Antibody (1:2000), Ubiquitin Antibody (1:1000), CISD1 Antibody (1:1000), CPT1α Antibody (1:1000), CYB5B Antibody (1:1000), HK1 Antibody (1:1000), MFN2 Antibody (1:1000), VDAC Antibody (1:1000) and Parkin Antibody (1:1000) in 5% BSA (TBS-Tween). Prepare phospho-Parkin Antibody (1:2000) in 5% milk (TBS-Tween). To avoid non-specific signal, it is recommended to preincubate phospho-Parkin antibody with a membrane for 2 days before using it.
1h
Remove primary antibody and wash 3 times with 0.1%TBS-Tween for 00:10:00 .
10m
Add secondary antibodies, HRP-conjugate for 01:00:00 at Room temperature diluted 1:5000 in 1% BSA (0.1% TBS-Tween). Use 1:10000 dilution in 1% BSA for Parkin antibody and 1:10000 dilution in 5% milk for and phospho-Parkin antibody.
1h
Remove secondary antibody and wash 3 times with 0.1%TBS-Tween for 00:10:00 .
10m
Develop signal using ECL western Blotting reagents and analysing with Chemidoc.
Note
Note: Depending on signal, film can be best for sensitivity. To improve detection of HK1 and VDAC ubiquitylation, it is recommended to develop signal using Super signal West Dura reagents.