Here we present a protocol to extract, resolve and identify mitochondrial supercomplexes which minimizes exposure to detergents and Coomassie Blue. This protocol offers an optimal balance between resolution, and preservation of enzyme activities, while minimizing the risk of losing labile protein-protein interactions.
Complexes of the oxidative phosphorylation machinery form supramolecular protein arrangements named supercomplexes (SCs), which are believed to confer structural and functional advantages to mitochondria. SCs have been identified in many species, from yeast to mammal, and an increasing number of studies report disruption of their organization in genetic and acquired human diseases. As a result, an increasing number of laboratories are interested in analyzing SCs, which can be methodologically challenging. This article presents an optimized protocol that combines the advantages of Blue- and Clear-Native PAGE methods to resolve and analyze SCs in a time-effective manner. With this hybrid CN/BN-PAGE method, mitochondrial SCs extracted with optimal amounts of the mild detergent digitonin are exposed briefly to the anionic dye Coomassie Blue (CB) at the beginning of the electrophoresis, without exposure to other detergents. This short exposure to CB allows to separate and resolve SCs as effectively as with traditional BN-PAGE methods, while avoiding the negative impact of high CB levels on in-gel activity assays, and labile protein-protein interactions within SCs. With this protocol it is thus possible to combine precise and rapid in gel activity measurements with analytical techniques involving 2D electrophoresis, immuno-detection, and/or proteomics for advanced analysis of SCs.
Mitochondria produce energy through oxidative phosphorylation, where respiratory complexes I-II-III-IV oxidize substrates and transfer electrons to oxygen, generating a gradient that allows phosphorylation of ADP by the ATP synthase (CV). In the past years, extensive studies have shown that respiratory chain complexes are not solely incorporated in a linear way in the inner mitochondrial membrane, but are also organised into supercomplexes (SCs) arrangements1,2. In mammalian mitochondria, SCs exist in varying stoichiometries: CI/CIII2/CIV1-4 (which is named the respirasome, and which is capable of NADH:O2 oxidoreduction in vitro)2, CI/CIII2, and CIII2/CIV1-2 3,4. Furthermore, respiratory complexes are distributed under different ratios between their free form and SCs arrangements. Therefore, it is estimated that 85%–100% of CI, 55%–65% of CIII, and 15%–25% of CIV are found in SCs4. These supramolecular structures are thought to decrease ROS production, stabilize or assist in the assembly of individual complexes, regulate respiratory chain activity, and prevent protein aggregation in the protein rich inner mitochondrial membrane5,6,7,8. Their remodelling ability upon variation in energy demand and their importance in the pathogenesis of diseases is being investigated in several labs3,7,9,10,11,12,13,14. Studies have demonstrated that pathological changes in SCs assembly are present in a variety of disorders, including, but not limited to, genetic defect in cardiolipin synthesis15, heart failure16, ischemia-reperfusion17, diabetes12, and aging18.
Native electrophoresis and immunodetection are widely used in SCs studies to resolve OXPHOS complexes quaternary arrangements2,19,20,21. Native electrophoresis can further be combined with specific in gel activity assays or 2D-SDS PAGE to enable precise molecular determination of the various SCs assemblies1,19. The ability to study SCs is critically dependent on the extraction conditions, including type and concentration of detergent used, ionic strength and pH, as well as on electrophoretic migration conditions, which comprise buffer composition, presence of CB, gel size, and acrylamide percentage2.
Protocols and SCs band resolution vary greatly among papers, making comparison between studies difficult and adaptation of methods challenging22. Therefore, this paper proposes a robust and optimal protocol to extract SCs from isolated mitochondria of different sources with the non-ionic detergent digitonin, and to resolve high molecular weight SCs bands. The optimized detergent concentration, composition of the extraction buffer, and the absence of Coomassie Blue in sample preparation minimize disruption of protein complexes. This protocol (see Figure 1 for an overview) combines CN-PAGE and BN-PAGE for optimal SCs assemblies resolution on large gel, and is compatible with in-gel activity assays allowing better visualization of reactive bands, along with the use of immunodetection for a detailed analysis SCs arrangements and composition.
1. SC Extraction
2. Gradient gel Casting and Electrophoresis
3. In-gel Activity for Complexes I, II, IV and CV
4. Immunoblotting
5. Analysis
Figure 2 shows results from a digitonin titration experiment aimed to identify the proper amount of digitonin required for the extraction of SCs. This amount will vary depending on the tissue/cell type and whether the sample was frozen or not. For this experiment, a CIV in-gel activity was performed to visualize SCs isolated from fresh mouse liver mitochondria. Ratios from 2/1 to 10/1 g digitonin/g of protein were tested. The optimal amount of digitonin for this sample is 4 g/g, as it provides a good resolution of monomeric CIV, and high molecular weight SCs. At a lower ratio, bands are not clear and resolve into a smear during electrophoresis, whereas the use of higher ratio of digitonin leads to disruption of high molecular weight SC.
Figure 3 and Figure 4 show the results of a complete experiment performed on a preparation of mouse liver mitochondria extracted with 4 g digitonin /g protein. Proteins were separated using hybrid BN/CN-PAGE, standard BN-PAGE, or CN-PAGE. All three gels were casted at the same time and lanes were loaded with replicates of the same sample. Following electrophoresis, individual lanes were cut and processed for in gel activity measurement (CI, CII, CIV and CV on Figure 3) and immunoblotting (CI, CII, CIII, CIV, CV on Figure 4).
Addition of CB either momentarily in cathode buffer (i.e. hybrid CN/BN-PAGE) or in sample and cathode buffer throughout electrophoresis (i.e. BN-PAGE), considerably improves the mobility and resolution of SC bands, and individual respiratory complexes compared to CN-PAGE (Figure 3). Bands are easily distinguishable with the hybrid technique or BN-PAGE after in-gel activity for CIV, whereas in the same sample resolved by CN-PAGE, SCs and monomeric CIV reactive bands cannot be identified.
Figure 3 and Figure 4 show that the resolution and banding pattern of OXPHOS monomers and supramolecular assemblies is qualitatively comparable between hybrid CN/BN-PAGE and BN-PAGE. However, notable differences exist. First, the electrophoretic mobility of OXPHOS complexes is slightly reduced when proteins are separated using hybrid CN/BN-PAGE conditions vs standard BN-PAGE, due to reduced amount of CB. This mobility shift is greater for CIV monomers, followed by CV monomers, and CI (Figure 3 and Figure 4). Second, the blue background is lower in the hybrid CN/BN-PAGE compared to BN-PAGE (Figure 3, left lanes). As a result, high background levels following BN-PAGE completely masks the in-gel activity staining for CII, and enhances the background noise associated with the activity of CIV dimers (Figure 3). Third, the activity of CV is higher when samples are run under hybrid CN/BN-PAGE conditions compared to BN-PAGE (Figure 3), due to the reduced amount of CB, which is known to interfere with CV catalytic activity.26 CN/BN-PAGE also allows better preservation of CV supramolecular assemblies, as shown by a greater proportion of total CV activity being associated with CV dimers (Figure 3). Moreover, CV oligomers are visible under CN/BN-PAGE, while they are completely dissociated under BN-PAGE conditions. Interestingly, distinct bands displaying CV activity are also observed between CV monomers and dimers, when samples are run under CN/BN-PAGE (Figure 2).
Figure 5 shows a representative analysis of OXPHOS complex distribution in supramolecular assemblies. The image shows CI in gel activity of samples obtained from 4 distinct healthy mouse liver mitochondria preparations. Densitometry analysis allows to measure the area under the curve of CI-reactive bands, and to present the relative distribution of C1 activity in the monomeric (I1) and supramolecular forms (I1III2, I1III2IV1, I1III2IVn). Similar analysis can be performed following immunoblot.
Figure 1: Assay workflow. Please click here to view a larger version of this figure.
Figure 2: Digitonin titration to extract supercomplexes from fresh mouse liver mitochondria. This example shows aliquots of mouse liver mitochondria, isolated from one animal that was treated with increasing amounts of digitonin to extract respiratory supercomplexes. Samples were then resolved by hybrid CN/BN PAGE, and in-gel activity of CIV was determined. CIV1: complex IV monomers; CIV2: Complex IV dimers; SC: supercomplexes. Please click here to view a larger version of this figure.
Figure 3: In-gel activity of OXPHOS complexes following hybrid CN/BN-PAGE, BN-PAGE or CN-PAGE. Liver mitochondria isolated from one mouse were treated with digitonin (4 g/g ratio digotonin/protein) to extract respiratory supercomplexes. Aliquots of this sample were then loaded on multiple wells in three distinct gels and submitted to CN/BN-PAGE, BN-PAGE or CN-PAGE. Each replicate lane within each gel was then cut and immediately used for in-gel activity assays (labeled CI, CII, CIV and CV). One lane was used as control to show background (labeled BG) staining with Coomassie Blue. OXPHOS complexes and supramolecular assemblies are identified using the standard nomenclature, with numbers in indices indicating the molecular stoichiometry of each OXPHOS complex. It should be noted that the position of CIII-containing supramolecular assemblies is based on immunodetection since in-gel activity for CIII was not performed in this particular experiment. Please click here to view a larger version of this figure.
Figure 4: Immunoblot analysis of OXPHOS complexes following hybrid CN/BN-PAGE or BN-PAGE. Replicates from the experiments described in the Figure 3 legend were electro-transferred on a single membrane. After transfer, individual lanes were cut and incubated with specific antibodies recognizing CI, CII, CIII, CIV, and CV. OXPHOS complexes and supramolecular assemblies are identified using the standard nomenclature, with numbers in indices indicating the molecular stoichiometry of each OXPHOS complex. Please click here to view a larger version of this figure.
Figure 5: Quantification of CI distribution in monomeric and supramolecular assemblies. (A) CI in-gel activity determined following Hybrid CN/BN-PAGE of in liver mitochondria SC extracts obtained from 4 mice. (B) Densitograms obtained using ImageJ’s Gel Analysis Tool showing distinct peaks corresponding to CI monomers (I1) and various CI-containing supramolecular complexes (I1III2, I1III2IV1, and I1III2IVn). (C) quantification of the relative distribution of C1 activity. The data represent mean and SEM of the 4 mice. Please click here to view a larger version of this figure.
Digitonin/protein ratio (g/g) | 2 g/g | 4 g/g | 6 g/g | 8 g/g |
Volume of extraction buffer (µL) | 400 | 300 | 200 | 100 |
Volume of 10 % stock digitonin (µL) | 100 | 200 | 300 | 400 |
Total extraction buffer volume (µL) | 500 | 500 | 500 | 500 |
Table 1: Volumes required to extract SCs from 5 mg of mitochondrial proteins using various digitonin/protein ratios.
Compound | Final Concentration |
EDTA, pH 7.5 | 1 mM |
HEPES | 30 mM |
Potassium Acetate | 150 mM |
Glycerol | 12% |
6-aminocaproic Acid | 2 mM |
Table 2: SC extraction buffer (final concentrations). Keep at 4 °C for a maximum of 3 months.
Compound | Final Concentration |
3X Gel Buffer: Aliquot and keep at -20 °C, pH 7.5 | |
Imidazole/HCl pH-7.0 | 75 mM |
6-aminocaproic Acid | 1.5 M |
Acrylamide Buffer: Aliquot and keep at -20 °C | |
Acrylamide | 99.5% |
Bis-Acrylamide | 3% |
Table 3: Gel stock buffers.
For 2 gels: | 4% (60 mL) | 12% (60 mL) | Stacking (4%) (25 mL) |
3X Gel Buffer | 19.8 mL | 19.8 mL | 8.25 mL |
Acrylamide Buffer | 4.8 mL | 14.4 mL | 2 mL |
H2O | 35 mL | 13.1 mL | 14.6 mL |
Glycerol | - | 12 mL | - |
APS 10% | 360 μL | 60 μL | 150 μL |
TEMED | 24 μL | 12 μL | 10 μL |
Table 4: 4%–12% gradient gel.
Compound | Final Concentration |
Anode Buffer: Keep at 4 °C, pH 7.5 | |
Imidazole | 25 mM |
Cathode Buffer: Keep at 4 °C, pH 7.5 | |
Tricine | 50 mM |
Imidazole | 7.5 mM |
With or without Coomassie Blue (G250) | 0.022% |
Table 5: Electrophoresis buffers.
Compound | Final Concentration |
Complex I activity Buffer: prepare fresh in 5 mM TRIS-HCl pH 7.4 | |
Nitrotetrazolium blue | 3 mM |
NADH | 14 mM |
Complex II activity Buffer: prepare fresh in 5 mM TRIS-HCl pH 7.4 | |
Succinate | 20 mM |
PMSF | 0.2 mM |
Nitrotetrazolium blue | 3 mM |
Complex IV activity Buffer: prepare fresh in 50 mM Na-Phosphate pH 7.2 | |
Cytochrome C | 0.05 mM |
Diaminobenzidine | 2.3 mM |
ATPsynthase activity Buffer: prepare fresh in water, adjust pH to 8 with KOH | |
Glycine | 50 mM |
MgCl2 | 5 mM |
HEPES | 50 mM |
CaCl2 | 30 mM |
ATP | 5 mM |
Table 6: In-gel activity assay buffers.
Compound | Final Concentration |
Transfer Buffer | |
Tris Base | 25 mM |
Glycine | 192 mM |
SDS | 4% |
Methanol | 20% |
TBST | |
Tris Base | 20 mM |
NaCl | 137 mM |
Tween 20 | 0.1% |
Table 7: Immunoblotting buffers.
Complex | Subunit | Clone |
I | NDUFA9 | 20C11B11B11 |
II | SDHA | 2E3GC12FB2AE2 |
III | UQCRC2 | 13G12AF12BB11 |
IV | COX4 | 1D6E1A8 |
V | ATPB | 3D5 |
Table 8: Antibodies used for immunoblotting to detect respiratory chain SC. See Table of Materials for companies and lot numbers.
Mitochondrial supercomplexes are being actively studied to elucidate their physiological role, and their importance in the pathogenesis of numerous human diseases, whether they are acquired or genetic mitochondrial diseases3,7,9,10,11,12,13,14. In order to obtain reliable results, several aspects need to be considered. This protocol has been tested with mouse liver mitochondria, mouse skeletal muscle mitochondria (results not shown), rat heart mitochondria, and human fibroblast mitochondria (results not shown), but could certainly be adapted to other sources of isolated mitochondria. The method optimally combines various aspects of BN and CN-PAGE protocols, which allow to reduce exposure to detergents and anionic compounds to a minimum compared to published protocols20,27,28.
Sample preparation
Sample preparation represents a crucial step for successful separation of SCs. Buffer composition should be carefully selected in order to achieve proper solubilization of proteins and proteins assemblies, while preserving as much as possible their functional and structural integrity. Ionic strength and pH of the extraction buffer are two important factors to consider. Salt concentrations that are too low (< 50 mM K-Acetate or NaCl) will result in poor solubilization of proteins in presence of non-ionic detergents, while salt concentrations above 500 mM will promote protein stacking/aggregation, and precipitation of CB and proteins29. SCs should therefore be extracted using buffers at near physiological ionic strength. With regards to pH, the use of a near physiological pH is recommended.
Detergent type and detergent/protein ratio are also critical for optimal SC extraction. For maximal preservation of native SCs, digitonin is preferred26. As shown in the present protocol and other published methods23,30,31,32, this mild detergent preserves the supramolecular composition of multiple SC assemblies, and the dimeric and oligomeric structure of ATPsynthase (Figure 3 and Figure 4). Titration of the samples of interest with various amounts of digitonin is critical in order to identify the conditions that allow optimal solubilization, while preserving enzyme activity and physiological protein interactions. Titration should be performed with ratios ranging between 2 and 8 g/g26. Optimal results for liver, skeletal muscle and cardiac mitochondria are respectively obtained with 4, 5, and 6 g digitonin/g protein. It should be noted that digitonin can be replaced by Triton X-100, which under optimal conditions results in similar migration and SC composition as those observed with digitonin2. However, this detergent should be used with caution, since relatively small increase in the detergent/protein ratio (e.g., from 1 to 1.5 g/g) can result in a complete dissociation of SCs assemblies2, which can result in experimental inconsistencies. After extraction, samples are traditionally supplemented with Coomassie Blue to give proteins a charge when applied to the gel, except for traditional CN-PAGE20,26. In order to minimize protein exposure to Coomassie blue and potential dissociation of labile proteins, samples are not supplemented with Coomassie blue in this protocol.
Electrophoresis
Both CN-PAGE and BN-PAGE have been used to study mitochondrial OXPHOS complexes, each of them having distinct advantages and limitations. The milder conditions used under CN-PAGE (mainly the absence of CB, which has a detergent-like effect), allows better preservation of ATP synthase in-gel activity, and limits the dissociation of labile proteins in high molecular weight SCs and ATP synthase assemblies26. However, the absence of the anionic dye CB in the protein extract and electrophoresis buffers causes the proteins to migrate based on their intrinsic charge and isoelectric point, which reduces the electrophoretic mobility of proteins within the gel26. Moreover, in the absence of CB, proteins with insufficient negative charge tend to aggregate, thus reducing the resolution of protein complexes in the gel20,26. To circumvent these limitations, the so-called high-resolution CN-PAGE has been developed by Wittig and Schragger20. In this protocol, sodium deoxycholate (DOC) and various mild non-ionic detergents (DDM, Triton X100) are added to the cathode buffer to keep membrane proteins solubilized and impose a negative charge shift on proteins, which results in a considerable improvement of resolution20.
A distinctive feature of the present hybrid CN/BN protocol is that a comparable resolution can be reached without these detergents. Momentary addition of CB to the cathode buffer at the beginning of the electrophoresis is sufficient to limit protein aggregation and enhance mobility in the gel (Figure 3 and Figure 4). As a result, this hybrid technique enables excellent resolution of distinct SC assemblies and very low or no exposure to detergents. The presence of low amounts of CB also allows better preservation of CV activity, improved preservation of dimeric and oligomeric CV assemblies (Figure 3 and Wittig and Schägger 200526), and a reduction of the blue background noise that can hinder the quantification of in gel activities, particularly for CII and CIV (Figure 2). Moreover, the absence of CB in the protein extract limits the disruption of labile protein interactions within SCs. For example, physical association of the ATP synthase with ANT to form the synthasome 33 or with Cyclophilin-D to regulate PTP opening 34 are better seen in absence of CB. Momentary exposure to CB during electrophoresis only may therefore be useful to reveal novel protein interactions within SCs. Overall, this hybrid CN/BN-PAGE protocol thus allows to combine precise and rapid in gel activity measurements with analytical techniques involving 2D electrophoresis, immuno-detection and/or proteomics for advanced analysis of SCs. It should be noted that with the growing interest for SCs, an increasing number of studies use small 10 x 10 cm gels for native PAGE. While this approach may be sufficient to identify gross changes in the abundance SC assemblies, the lower separation capacity of small gels is likely limited to resolve subtle rearrangements or to cut distinct bands for proteomic analysis. Moreover, several studies using smaller gels have reported that the respirasome migrates at the same size as the ATPsynthase dimer, making it difficult to dissociate them22. Therefore, the use of large gels should be favored.
The authors would like to thank Jenna Rossi for technical assistance, and Dr. Mireille Khacho, Dr. David Patten and Dr. Ujval Anil Kumar for helpful discussion while developing this method. This work was funded by the Canadian Institutes of Health Research (CIHR) and the National Sciences and Engineering Council of Canada (NSERC). AC is a recipient of Doctoral Award - Frederick Banting and Charles Best Canada Graduate Scholarships (CIHR).
Name | Company | Catalog Number | Comments |
3,3'-Diaminobenzidine tetra-hydrochloride hydrate | Sigma | D5637 | |
6-amino caproic acid | sigma | A2504 | |
Acrylamide | Sigma | A3553 | |
Adenosine 5'-triphosphate disodium salt hydrate | sigma | A3377 | |
Anti-ATPB antibody [3D5] - Mitochondrial Marker | Abcam | ab14730 | Lot number GR3174539-12, RRID AB_301438 |
Anti-SDHA antibody [2E3GC12FB2AE2] | Abcam | ab14715 | Lot number GR3235943-1, RRID AB_301433 |
Anti-UQCRC2 antibody [13G12AF12BB11] | Abcam | ab14745 | Lot number GR304308-3, RRID AB_2213640 |
Bis N,N'-Methylene-Bis-Acrylamide | Biorad | 1610201 | |
Brilliant Blue G | Sigma | 27815 | |
COX4 Monoclonal Antibody (1D6E1A8) | Invitrogen | 459600 | Lot number TI2637158, RRID AB_2532240 |
Cytochrome c from equine heart | sigma | C7752 | |
Digitonin | Sigma | D141 | |
Fisherbrand FH100M Multichannel Peristaltic Pumps | Thermo Fisher | 13-310-660 | |
Imidazole | Sigma | I0250 | |
Inner Glass Plates. Pkg of 2, 20 x 20 cm, glass plates for 20 cm PROTEAN II xi and PROTEAN II XL electrophoresis cells, use with outer plate | Biorad | 1651823 | |
Model 485 Gradient Former. 40-175 ml acrylamide gradient former, includes valve stem and tubing kit, for use with Mini-PROTEAN multi-casting chamber systems | Biorad | 1654120 | |
NDUFA9 Monoclonal Antibody (20C11B11B11) | Invitrogen | 459100 | Lot number TD2536591, RRID AB_2532223 |
Nitrotetrazolium Blue chloride | Sigma | N6639 | |
Outer Glass Plates. Pkg of 2, 22.3 x 20 cm, glass plates for 20 cm PROTEAN II xi and PROTEAN II XL electrophoresis cells, use with inner plate | Biorad | 1651824 | |
Phenylmethanesulfonyl floride | Sigma | P7626 | |
Powerpack 1000 | Biorad | Serial Number 286BR 07171 | |
PROTEAN II Sandwich Clamps. Pkg of 2, clamps for running gels, for 20 cm PROTEAN II xi electrophoresis cell, 1 left and 1 right | Biorad | 1651902 | |
PROTEAN II xi Cell. Large format vertical electrophoresis cell, 16 x 20 cm gel size, 4 gel capacity, spacers and combs | Biorad | 1651811 | |
PROTEAN II xi Comb. Pkg of 1, 15-well, 1.5 mm, comb for PROTEAN II xi electrophoresis cell | Biorad | 1651873 | |
PROTEAN II xi Multi-Gel Casting Chamber. Multi-gel casting chamber, 20 x 20 cm gel size, for up to ten 1.5 mm thick gels, includes sealing plate, gasket, separation sheets, acrylic blocks, PROTEAN II XL alignment cards | Biorad | 1652025 | |
PROTEAN II xi Spacers. Pkg of 4, 1.5 mm, spacers for 20 cm PROTEAN II xi electrophoresis cell | Biorad | 1651849 | |
SCIENCEWARE Utility Bags (10 x 12") 4 mil, Bel-Art, Box of 100 | VWR | 11215-388 | |
Thick Blot Paper. Pkg of 25 sheets, 15 x 20 cm, absorbent filter paper, for use with Trans-Blot cassette | Biorad | 1703956 | |
Trans-Blot Cell With Plate Electrodes and Super Cooling Coil. Transfer cell and cooling coil (#170-3912), includes 2 gel holder cassettes, buffer tank, lid with cables, fiber pads, 1 pack blot paper | Biorad | 1703939 |
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