Jan 27, 2025
Mito-IP: Immunopurification of Mitochondria from MitoTag Cultured Cells
- Christos Themistokleous1,
- Miratul M K Muqit1
- 1Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Str, Dundee DD1 5EH, UK
Protocol Citation: Christos Themistokleous, Miratul M K Muqit 2025. Mito-IP: Immunopurification of Mitochondria from MitoTag Cultured Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj9q8blk5/v1
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: January 03, 2025
Last Modified: January 27, 2025
Protocol Integer ID: 118215
Keywords: Immunoprecipitation, Mito-Tag, Homogenisation
Abstract
Cell organelles represent a minor fraction of the total cellular content, making whole-cell profiling inadequate for monitoring changes in the mitochondrial proteome, metabolome, and lipidome. Traditional techniques for purifying mitochondria have inherent limitations, often compromising organelle purity, isolation time, or viability. Additionally, the components of conventional organellar isolation buffers, such as sucrose, can interfere with mass spectrometry (MS) profiling. To overcome these challenges, a novel method called 'Mito-IP,' was developed which facilitates the rapid immunopurification of pure and intact mitochondria. This method enables mitochondrial isolation within 10 minutes and supports various downstream applications, including immunoblotting, proteomic, metabolomic, and other -omic analyses. Following the success of the Mito-IP method, the same epitope-tagged concept is being extended to the isolation of other organelles, including lysosomes (Lyso-IP), Golgi (Golgi-IP), and peroxisomes (Peroxo-IP). The following optimised protocol details the immunoprecipitation of mitochondria from cultured cells. The same steps apply to the immunopurification of other organelles when the HA-epitope tag is present on the organelle of interest.
Guidelines
INTRODUCTION:
Cell organelles represent a minor fraction of the total cellular content, making whole-cell profiling inadequate for monitoring changes in the mitochondrial proteome, metabolome, and lipidome. Traditional techniques for purifying mitochondria have inherent limitations, often compromising organelle purity, isolation time, or viability. Additionally, the components of conventional organellar isolation buffers, such as sucrose, can interfere with mass spectrometry (MS) profiling.
To overcome these challenges, a novel method called "Mito-IP," was developed which facilitates the rapid immunopurification of pure and intact mitochondria. This method enables mitochondrial isolation within 10 minutes and supports various downstream applications, including immunoblotting, proteomic, metabolomic, and other -omic analyses (Figure 1). The method employs a chimeric protein comprising of three HA epitope tags fused to the outer mitochondrial membrane protein OMP25 (rat, aa109-145). An LC/MS-compatible buffer (termed "KPBS") was also developed containing only KCl and KH₂PO₄, significantly improving performance and mitochondrial viability.
Following the success of the Mito-IP method, the same epitope-tagged concept is being extended to the isolation of other organelles, including lysosomes (LysoIP), Golgi (GolgiIP), and peroxisomes (PeroxoIP). The following optimised protocol details the immunoprecipitation of mitochondria from Mito-Tag cultured cells. The same steps apply to the immunopurification of other organelles when the HA-epitope tag is present on the organelle of interest.
Fig. 1. Schematic of the Mito-IP method. Cells are collected and homogenized using a cell homogenizer in KPBS buffer. After the release of intact organelles from the cells, the homogenate is incubated with anti-HA magnetic beads for 5 minutes, allowing the beads to bind to the mitochondria. Using a magnetic separator, the mitochondria-bound beads are drawn to the side of the tube. Following three washes, an appropriate lysis buffer is added for downstream analysis.
Materials
MATERIALS & REAGENTS
- Cell lines:
- HEKInvitrogen™ 293FT Cell LineInvitrogen - Thermo FisherCatalog #R70007
- The cell line of interest for introducing the Mito-Tag.
- Plasmids:
- Control-Tag construct: pLJC5-KOZAK-3HA-Empty (DU70022 available at MRCPPU depository at MRCPPUreagents@dundee.ac.uk)
- Mito-Tag construct: pMXs-3XHA-EGFP-OMP25addgeneCatalog #83356 or available at MRCPPU depository at MRCPPUreagents@dundee.ac.uk)
- pVSVG - lentivirus envelope plasmid: Lenti-X HTX packaging system (Clonetech, #631247)
- pGag/Pol - Lentivirus Gag/Pol plasmid: Lenti-X HTX Packaging system (Clonetech, #631247)
- pVSVG - retrovirus envelope plasmid: Addgene plasmid #8454)
- pGag/Pol - retrovirus plasmid: Addgene plasmid #8449)
- Growth Media:
- DMEM (Gibco™ #11960-085)Gibco - Thermo FischerCatalog #11960085
- 10% Fetal Bovine SerumMerck MilliporeSigma (Sigma-Aldrich)Catalog #F7524
- 1% Penicillin-StreptomycinGibco - Thermo FisherCatalog #15140122
- 1% L-Glutamine Gibco - Thermo FisherCatalog #25030024
- Selection Media: Growth Media with 2μg/ml Puromycin dihydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #P9620
- Transfection media: Opti-MEM Gibco - Thermo FisherCatalog #31985062 .
- PEI MAX® - Transfection Grade Linear Polyethylenimine Hydrochloride (MW 40000)Polysciences, Inc.Catalog #24765-1
- Polybrene Infection/Transfection reagentMerck MilliporeSigma (Sigma-Aldrich)Catalog #TR-1003-G
- DPBS no calcium no magnesiumGibco - Thermo FischerCatalog #14190169
- KPBS Buffer: 136mM KCl, 10mM KH2PO4. Adjust to pH 7.25 with KOH and filter (0.22μm conring). On the day of use, add cOmplete™ EDTA-free Protease Inhibitor CocktailRocheCatalog #11873580001 and Roche PhosSTOP tablet (#04906837001).
- Lysis Buffer
- MS grade water (Fisher, 11947199)
- Pierce BCA protein assayThermo ScientificCatalog #23227 , lot# VA294738)
- NuPAGE 4xLDS sample buffer (Invitrogen, #1941674)
- 2-mercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M6250
EQUIPMENT
- Cell homogeniser (Isobiotec).
- Orbiter (The Belly Dancer Shaker; IBI Scientific, #BDRAA115S)
Equipment
The Belly Dancer Shaker (Orbiter)
NAME
The Belly Dancer®
BRAND
BDRAA115S
SKU
LINK
- DynaMag™ Magnet. (Thermofisher scientific, #12320D or #12321D).
Equipment
Magnet
NAME
DynaMag™-2 Magnet
TYPE
Thermo Fisher
BRAND
12321D
SKU
LINK
- Microcentrifuge (VWR Micro Star 17R. S/N 42209232, # 521-1647).
Equipment
Microcentrifuges, Micro Star 17R (VWR #521-1647)
NAME
Microcentrifuges
TYPE
Micro Star 17R
BRAND
521-1647
SKU
LINK
- Bioruptor NGS Sonication System (UCD-600).
- Incubator with FPI-sensor system and display controller MB1 (BINDER GmbH. Model: CB150. Power Output: 1.40kW, 230V, 6.1 Amp).
CONSUMABLES
- Pierce™ Anti-HA Magnetic BeadsThermo FisherCatalog #88837
- 10cm and 15cm tissue culture Petri Dishes (ThermoFisher, #172931 and 16838).
- Syringe filter (0.45µm. Sartorius, #ST16537-Q)
- Syringes (10ml) (Medicina, #19111004).
- Fisherbrand™ Cell LiftersThermo Fisher ScientificCatalog #08-100-240
- Eppendorf Tubes® 3810XEppendorfCatalog #0030125150
- 15 ml centrifuge tubes greiner bio-oneCatalog #188271
- 50 ml centrifuge tubes greiner bio-oneCatalog #227261
- TipOne bevelled 1000μl, 200µl and 20µl pipette tips (Starlab).
Generation of MitoTag or ControlTag Retrovirus (Timing: 3d)
Generation of MitoTag or ControlTag Retrovirus (Timing: 3d)
2d 0h 35m
2d 0h 35m
Note
! Caution: Work under sterile conditions in a category 2 biological safety hood.
Plate 2 10cm Petri dishes of HEK293FT cells to 60% confluency in Growth media (one dish to generate the MitoTag retrovirus and the other one the ControlTag lentivirus).
Note
- Different batches of HEK293T cells vary in their ability to produce high-tier viruses and can be sensitive to overcrowding, media changing and trypsin digestion.
- An alternative ControlTag for the organelleIP can be the: 3xMyc-EGFP-OMP25.
- The presence of GFP in the MitoTag can help with immunofluorescence (IF) and cell sorting experiments. If not needed, the MitoTagLite construct (3xHA-OMP25) can be used instead.
MitoTagLite construct: pBabeD-3XHA-OMP25 (DU71376, available at MRCPPU depository at MRCPPUreagents@dundee.ac.uk).
Prepare a transfection mix for each plate in a 1.5ml Eppendorf tube.
| A | B | |
| Component | per 10cm dish | |
| pGag/Pol plasmid | 3.8 µg | |
| pVSVG plasmid | 2.2 µg | |
| Mito-Tag or ControlTag plasmid | 6 µg | |
| Opti-MEM | 300 µl |
Note
Plasmids were purified using a NucleoBond Xtra Midi kit (ref 740410.50) following the manufacturer’s protocols and instructions.
Prepare PEI mixture in a 1.5ml Eppendorf tube.
| A | B | |
| Component | per 10cm dish | |
| PEI (1mg/ml, dissolved in dH2O) | 40 µl | |
| Opti-MEM | 600 µl |
Incubate each mixture from steps 2 and 3 separately for 00:05:00 at Room temperature .
5m
Add 300 µL PEI Mixture (step 3) to the MitoTag and ControlTag tube (step 2).
Mix and incubate each mixture at Room temperature for 00:30:00 . During this time, carefully replace the medium of each plate of HEK293T cells with 10 mL of fresh Growth Media.
30m
Add each mixture dropwise into a HEK293FT plate and gently swirl to mix.
Incubate cells at 37 °C for 24:00:00 .
1d
Replace the medium of each dish with 10 mL fresh Growth Media and incubate cells at 37 °C for a further 24:00:00 .
1d
Harvest the virus by collecting the medium and passing it through a 0.45µm syringe filter. This infection medium can be used immediately or stored at -80 °C .
Note
Critical step: The virus can be stored at -80 °C for years. Because viruses are sensitive to freeze-thaw cycles, multiple aliquots of 3 mL can be made.
Note
Note: If more than 10 mL of infection media will be needed, this procedure can be scaled up by plating more than one dish per condition. In the end, infection media from different plates of the same condition can be combined and aliquoted before freezing down. Alternatively, after collecting the infection media, add 10 mL fresh Growth Media to the plates and recollect after 24h (second harvest).
Generation of Epitope-Tagged Mitochondria (Transduction and Selection) (Timing: 2d)
Generation of Epitope-Tagged Mitochondria (Transduction and Selection) (Timing: 2d)
2d
2d
Note
! Caution: Work under sterile conditions in a category 2 biological safety hood.
Mix 3 mL of infection media with 7 mL fresh Growth Media.
Add Polybrene to the Mix using a stock of 10 µL (dissolved in MilliQ water and sterile filtered) to a final concentration of 10 µL .
Gently add 10 mL of the mix to a 10cm plate of cells already at 60% confluency.
Note
Mouse and human cell types can be infected with this media (e.g., mouse embryonic fibroblasts; MEF, A549, etc). The amount of virus needed per infection is dependent on the titer of the virus and the infectability of the cell line being used. The expression levels of the MitoTag can affect the yield and purity of mitochondrial capture. It is recommended to use different dilutions of virus:media (e.g. 2:8, 4:6, 6:4) and proceed with the ones that show optimal Mito-IP results by biochemical analysis. Lower expression of the construct is generally better, achieving amounts of mitochondrial capture similar to those of higher expressing systems while substantially reducing contamination with organelles such as the peroxisome. It is important to note that mitochondria can directly interact with certain organelles, such as the endoplasmic reticulum and peroxisomes, in living cells; the strength of these interactions may vary among cell types and lead to different amounts of contamination in immunopurified mitochondria, but there should generally be an enrichment of mitochondrial markers in the IP lysates as compared with whole-cell lysates. If the used construct contains EGFP, FACS can be used to sort and obtain cells with an appropriate amount of EGFP signal (preferably the ones expressing low to mid-levels of EGFP).
Incubate at 37 °C for 24:00:00 .
1d
Change media to Growth Media and incubate at 37 °C for another 24:00:00 .
1d
To select cells stably expressing the MitoTag or ControlTag, replace media with 10 mL freshly prepared selection media. After 24-48h, there will be observable dead cells in the cultures.
Note
Different cell lines have varying sensitivities to antibiotics, and it is important to empirically determine the working concentration needed to completely kill uninfected cells (kill curve). Puromycin selection requires around 48h while blasticidin selection can take up to 7 days.
Change Selection Media every 48h for 4-7 days (depending on the efficiency of the transduction process). At this stage cells stably expressing the MitoTag or ControlTag should be at 90% confluency.
Note
If the cell colonies do not grow or do not grow fast enough, move the cells from a 10cm dish to a 6cm dish or optimise the infection/ virus production steps. Sometimes the transfection efficiency is high and there are no dead cells.
Split the cells into more dishes while maintaining them in selection Media. Cells can be frozen down and stored long-term in liquid nitrogen.
Note
Cells should be grown only in selection media, especially before freezing them down. The Selection Media can be replaced by Growth Media only when thawed to help them recover the first 24-48h or before an experiment.
Cell Preparation for MitoTag and ControlTag Immunoprecipitation (2-3 days prior to immunoprecipitation)
Cell Preparation for MitoTag and ControlTag Immunoprecipitation (2-3 days prior to immunoprecipitation)
Split cells stably expressing MitoTag or ControlTag in a 15cm Petri dish. Allow to grow near confluency (approximately 48h depending on the seeding density).
Note
Using a 15cm instead of a 10cm dish significantly increases the Mito-IP yield.
DAY OF THE IMMUNOPRECIPITATION Pre-clearing of Anti-HA Beads (Timing: 5 min)
DAY OF THE IMMUNOPRECIPITATION Pre-clearing of Anti-HA Beads (Timing: 5 min)
2m 30s
2m 30s
Resuspend beads by shaking the bottle until there is a homogeneous suspension.
Pipette 200 µL anti-HA bead slurry into a tube (100 µL per sample).
Place the tube onto a magnet for 00:00:30 and carefully remove the overlying solution.
30s
Remove the tube from the magnet, add 1 mL of cold KPBS and gently resuspend 3 times to disperse any clumps.
Repeat the wash step (22+23) 2 more times.
1m
- Place the tube onto a magnet for 00:00:30 and carefully remove the overlying solution.
- Remove the tube from the magnet, add 1 mL of cold KPBS and gently resuspend 3 times to disperse any clumps. (1/2)
30s
- Place the tube onto a magnet for 00:00:30 and carefully remove the overlying solution.
- Remove the tube from the magnet, add 1 mL of cold KPBS and gently resuspend 3 times to disperse any clumps. (2/2)
30s
After the last wash, resuspend the beads in 200 µL of KPBS, split it into 2 tubes of 100 µL (one for the Mito-IP and one for the Control-IP) and keep them On ice .
Note
The volume of beads used can be adjusted.
Cell Collection and Homogenisation (Timing: 10min)
Cell Collection and Homogenisation (Timing: 10min)
4m
4m
Place cells On ice and remove the media by pouring it into a container.
Wash the plates twice with 10 mL cold PBS (pour it in and out if the cells are well attached to increase the speed of the workflow. Alternatively use pipettes).
Aspirate all of the PBS and add 1 mL of KPBS.
Scrape the cells while On ice with a cell lifter.
Carefully transfer scrapped cells using a P1000 pipette into a 1.5ml (or 2ml) tube.
Spin down at 1000 x g, 4°C, 00:02:00 .
2m
Discard the supernatant and resuspend the pellet in 950 µL of KPBS.
Transfer 25 µL into a new tube (whole-cell input) and keep it On ice .
Move the cells in a 1ml syringe with a 21G needle and place it on the homogeniser.
Note
Wash, assemble and prechill the homogeniser beforehand. Insert in the stainless-steel block a ball that leaves a 10μm gap and screw the lids on tightly. Place it On ice and with the help of two 1ml syringes pass through the device 1ml of KBPS a couple of times (to fill the device with KPBS instead of water and remove any bubbles).
With the help of a second 1ml syringe pass the cells 20 times through the bore.
Note
Ensure you hold the syringes firmly while passing the homogenate through the device, as the build-up pressure can force the syringes out of the openings, resulting in the loss of the homogenate. If excessive pressure prevents the homogenate from passing through the bore, adjust the size of the ball used to leave a bigger gap and the number of passes.
Transfer the homogenate into a new 1.5ml tube.
Note
To collect the remaining sample in the device, inject some air into one of the openings using a syringe and collect the sample from the other opening with a second syringe.
Spin down at 2000 x g, 4°C, 00:02:00 .
Note
This step ensures the removal of the non-lysed cells, nuclei and debris such as cell membranes and leave the intact organelles in the supernatant.
2m
Move 25 µL to a new tube (IP input) and keep it On ice .
Carefully move the supernatant (700 µL -800 µL ) into the tube containing the prewashed beads. Ensure that the bead clumps are dispersed by gently pipetting up and down 3 times.
Note
Critical step: Be very careful not to collect any of the pellet as it will affect the purity of the IP. For some cell homogenates, the separation between the pellet and the supernatant is not very clear. In this case, collect less supernatant.
Note
Continue in the cold room until the detergent lysis step
Immunoprecipitation (Timing: 9min)
Immunoprecipitation (Timing: 9min)
5m 30s
5m 30s
Incubate the homogenate with the beads for 00:05:00 on an orbiter.
Note
- Incubation time can be adjusted.
- In the meantime, you can wash and prepare the cell homogeniser for the next sample. Open it from one side, remove the ball, wash it by passing some ultrapure water through it and close it. After the processing of the last sample, wash and clean each part with 70% (v/v) ethanol and leave them to dry before assembling the device back to avoid developing rust.
5m
Separate the beads by putting the tube on the magnet for 00:00:30 .
Note
Residual material left on the cap can be brought down with a pulse spin (< 1s) before placing the tubes on the magnet.
30s
Collect 25 µL of flow-through into a new tube (FT input) and keep it On ice .
Wash beads with 1000µl of cold KPBS 3 times.
Note
Move the beads to a new tube after every wash to minimise contamination from cell extracts sticking to the tube.
The isolated mitochondria on the beads can be either stored at -80 °C for later use or eluted off the beads using an appropriate lysis buffer:
- Immunoblot analysis
- Proteomic analysis
- Lipidomic analysis
- Metabolomic analysis
- Other
Elusion and Lysis (Timing: 25-45min)
Elusion and Lysis (Timing: 25-45min)
40m 30s
40m 30s
Incubate the beads with 50 µL lysis buffer for 00:10:00 .
10m
Place the tube on the magnet for 00:00:30 and collect the supernatant into a new tube. Repeat this step one more time to ensure total removal of the beads.
30s
For the input samples, add 100 µL of lysis buffer, resuspend and incubate for 00:20:00 .
20m
Leave samples oOn ice and continue with the next round of IP.
Spin down input samples at 13000 x g, 4°C, 00:10:00 and move supernatant into a new tube.
10m
For samples intended for immunoblot or proteomic analysis: Sonicate the IP and input samples for maximal protein extraction (15 cycles – 30sec on, 30sec off). Determine the protein concentration of the IP and inputs using a micro BCA Protein Assay Kit. Use 2 µL of undiluted IP samples and 2 µL of diluted (1:10) input samples. Quantification should be done in triplicate or duplicate.
Samples can be stored at -80 °C for future application.
For immunoblot analysis: samples are diluted into 4xLDS loading buffer supplemented with fresh 5% (by vol) 2-mercaptoethanol prior to analysis on SDS-polyacrylamide gel electrophoresis and immunoblot analysis. Loading 1 µg -2 µg of sample is enough to detect the mitochondrial markers (such as HSP60, CS, VDAC, CISD1, OPA1, MFN).
For sample preparation for proteomic analysis, use the protocol:
'Sample preparation for proteomic analysis of isolated mitochondria and whole-cell extracts'