This work was supported by grants from the American Heart Association Founder's Affiliate [12GRNT12060233] and the Strong Children's Research Center at the University of Rochester.

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R., Kuhlbrandt, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimer+ribbons+of+ATP+synthase+shape+the+inner+mitochondrial+membrane.">Dimer ribbons of ATP synthase shape the inner mitochondrial membrane.</a> EMBO J. 27 (7), 1154-1160 (2008).</li><li>Beutner, G., Eliseev, R. A., Porter, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+electron+transport+chain+activity+in+the+embryonic+heart+coincides+with+the+activation+of+mitochondrial+complex+1+and+the+formation+of+supercomplexes.">Initiation of electron transport chain activity in the embryonic heart coincides with the activation of mitochondrial complex 1 and the formation of supercomplexes.</a> PloS one. 9 (11), e113330 (2014).</li><li>Genova, M. L., Lenaz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Interplay+Between+Respiratory+Supercomplexes+and+ROS+in+Aging.">The Interplay Between Respiratory Supercomplexes and ROS in Aging.</a> Antioxid Redox Signal. 23 (3), 208-238 (2015).</li><li>Rosca, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+mitochondria+in+heart+failure:+decrease+in+respirasomes+and+oxidative+phosphorylation.">Cardiac mitochondria in heart failure: decrease in respirasomes and oxidative phosphorylation.</a> Cardiovasc Res. 80 (1), 30-39 (2008).</li><li>Kuter, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+within+mitochondrial+oxidative+phosphorylation+supercomplexes+and+membrane+viscosity+during+degeneration+of+dopaminergic+neurons+in+an+animal+model+of+early+Parkinson's+disease.">Adaptation within mitochondrial oxidative phosphorylation supercomplexes and membrane viscosity during degeneration of dopaminergic neurons in an animal model of early Parkinson's disease.</a> Biochim Biophys Acta. 1862 (4), 741-753 (2016).</li><li>Wittig, I., Karas, M., Schagger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+clear+native+electrophoresis+for+in-gel+functional+assays+and+fluorescence+studies+of+membrane+protein+complexes.">High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes.</a> Mol Cell Proteomics. 6 (7), 1215-1225 (2007).</li><li>Fiala, G. 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The authors have nothing to disclose.

A functional ETC is necessary for mitochondrial ATP generation. The complexes of the ETC are able to form two types of supercomplexes: the respirasomes (Cx-I, -III, and -IV)1 and the synthasomes (Cx-V)2. The assembly of each complex is required for an intact ETC, while the organization of the ETC into supercomplexes is thought to increase overall ETC efficiency5,22. How these supercomplexes assemble and disassemble ...

Mitochondrial energy in the form of ATP is not only essential for cell survival, but also for the regulation of cell death. The generation of ATP by oxidative phosphorylation requires a functional electron transport chain (ETC; Cx-I to IV) and mitochondrial ATP synthase (Cx-V). Recent studies have shown that these large protein complexes are organized into supercomplexes, called respirasomes and synthasomes1,2. It is challenging to analyze the assembly, dynamics, and activity regulation of these massive complexes and supercomplexes. While oxygen consumption measurements taken with an oxygen electrode and enzyme assays conducted using a spectrophotometer can give valuable information about ETC complex activity, these assays cannot provide information regarding the presence, size, and subunit composition of the protein complex or supercomplexes involved. However, the development of blue and clear native (BN and CN, respectively) PAGE3 has created a powerful tool for revealing important information about complex composition and assembly/disassembly and about the dynamic regulation of the supramolecular organization of these vital respiratory complexes under physiological and pathological conditions4.
The assembly of these complexes into higher-order supercomplexes appears to regulate mitochondrial structure and function5. For example, respirasome assembly increases the efficiency of electron transfer and the generation of the proton motive force across the mitochondrial inner membrane5. In addition, the assembly of synthasomes not only increases the efficiency of ATP production and the transfer of energy equivalents into the cytoplasm2, but it also molds the mitochondrial inner membrane into the tubular cristae6,7. Studies of supercomplex assembly during cardiac development in mouse embryos show that the generation of Cx-I-containing supercomplexes in the heart begins at about embryonic day 13.58. Others have shown that the amount of Cx-I-containing supercomplexes decreases in the heart due to aging or ischemia/reperfusion injuries9,10 or may play a role in the progression of neurodegenerative diseases11.
This protocol describes methods for native gel PAGE that can be used to investigate the assembly and activity of the ETC complexes and supercomplexes. The approximate molecular weight of mitochondrial supercomplexes can be assessed by separating the protein complexes in CN or BN polyacrylamide gels. CN PAGE also allows for the visualization of the enzymatic activity of all mitochondrial complexes directly in the gel (in-gel assays; IGA)12. This work demonstrates the activity of respirasomes by highlighting the ability of Cx-I to oxidize NADH through IGA and the presence of synthasomes due to the ATP-hydrolyzing activity of Cx-V by IGA. The multiple complexes and supercomplexes containing Cx-I and Cx-V can also be demonstrated by transferring the proteins onto nitrocellulose membranes and performing immunoblotting. The advantage of this approach is that BN or CN PAGE generally separates protein complexes based on their physiological size and composition; the transfer to a membrane preserves this pattern of bands. Analyzing protein complexes in a BN or CN PAGE can also be done using 2D-PAGE (see Fiala et al.13 for a demonstration) or by sucrose density centrifugation14,15. To further analyze a specific band, it can be excised from the BN PAGE, and the proteins from this protein complex can be purified by electroeluting them under native conditions. Native electroelution can be performed within a few hours, which could make a significant difference to the passive diffusion (as used in Reference 16) of proteins from a gel into the surrounding buffer.
In summary, these methods describe several approaches that allow for the further characterization of high-molecular-weight supercomplexes from mitochondrial membranes.

<table><tbody><tr><td>Protean II mini-gel chamber</td><td>Biorad</td><td>1658004</td><td>Complete set to pour and run mini-gel electrophoresis</td></tr><tr><td>Protean XL maxi-gel</td><td>Biorad</td><td>1653189</td><td>Complete set to pour and run maxi-gel electrophoresis</td></tr><tr><td>Gradient maker, Hoefer SG15</td><td>VWR</td><td>95044-704</td><td>Pouring mini-gel gradients</td></tr><tr><td>Gradient maker, maxi-gel</td><td>VWR</td><td>GM-100</td><td>Pouring maxi-gel gradients</td></tr><tr><td>Transfer kit</td><td>Biorad</td><td>1703930</td><td>Complete set to wet transfer of proteins onto membranes</td></tr><tr><td>Electroeluter model 422</td><td>Biorad</td><td>1652976</td><td>Electroelution of proteins from native or SDS PAGES</td></tr><tr><td>Glass plates</td><td>Biorad</td><td>1653308</td><td>Short plates</td></tr><tr><td>Glass plates</td><td>Biorad</td><td>1653312</td><td>Spacer plates</td></tr><tr><td>Glass plates</td><td>Biorad</td><td>1651823</td><td>Inner plates</td></tr><tr><td>Glass plates</td><td>Biorad</td><td>1651824</td><td>Outer Plates</td></tr><tr><td>Power supply</td><td>Biorad</td><td>1645070</td><td>Power supply suitable for native electrophoresis</td></tr><tr><td>ECL-Western&amp;nbsp;</td><td>Thermo Scientific</td><td>32209</td><td>Chemolumniscense substrate</td></tr><tr><td>SuperSignal-West Dura</td><td>Thermo Scientific</td><td>34075</td><td>Enhanced chemolumniscense substrate</td></tr><tr><td>Film/autoradiography film</td><td>GE Health care</td><td>28906845</td><td>Documentation of Western blots</td></tr><tr><td>Film processor CP1000</td><td>Agfa</td><td>NC0872640</td><td></td></tr><tr><td>Canon Power Shot 640&amp;nbsp;</td><td>Canon</td><td>NA</td><td>Taking photos to document gels, membranes and blots.</td></tr><tr><td>Canon Power Shot 640 Camera hood&amp;nbsp;</td><td>Canon</td><td></td><td>shielding camera for photos being taken on a light table</td></tr><tr><td>Acrylamide/bisacrylamide</td><td>Biorad</td><td>1610148</td><td>40% pre-mixed solution</td></tr><tr><td>Glycine</td><td>Sigma</td><td>G7403</td><td></td></tr><tr><td>SDS (sodium dodecyl sulfate)</td><td>Invitrogen</td><td>15525-017</td><td></td></tr><tr><td>Tris-base</td><td>Sigma</td><td>T1503</td><td>Buffer</td></tr><tr><td>Tricine</td><td>Sigma</td><td>T0377</td><td></td></tr><tr><td>Sodium deoxychelate</td><td>Sigma</td><td>D66750</td><td>Detergent</td></tr><tr><td>EDTA</td><td>Sigma</td><td>E5134</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S9378</td><td></td></tr><tr><td>MOPS</td><td>Sigma</td><td>M1254</td><td>Buffer</td></tr><tr><td>Imidazole</td><td>Sigma</td><td>I15513</td><td>Buffer</td></tr><tr><td>Lauryl maltoside</td><td>Sigma</td><td>D4641</td><td>Detergent</td></tr><tr><td>Coomassie G250</td><td>Biorad</td><td>161-0406</td><td></td></tr><tr><td>Aminohexanoic acid</td><td>Sigma</td><td>O7260</td><td></td></tr><tr><td>Native&amp;nbsp; molecular weight kit</td><td>GE Health care&amp;nbsp;</td><td>17-0445-01</td><td>High molecular weight calibraition kit for native electrophoresis.</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>NADH</td><td>Sigma</td><td>N4505</td><td></td></tr><tr><td>Nitroblue tetrazolium</td><td>Sigma</td><td>N6639</td><td></td></tr><tr><td>Tris HCl</td><td>Sigma</td><td>T3253</td><td></td></tr><tr><td>ATP&amp;nbsp;&amp;nbsp;</td><td>Sigma</td><td>A2383</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>Lead(II) nitrate (Pb(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;):</td><td>Sigma</td><td>228621</td><td></td></tr><tr><td>Oligomycin</td><td>Sigma</td><td>O4876</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>Ponceau S</td><td>Sigma</td><td>P3504</td><td></td></tr><tr><td>anti-ATP5A</td><td>Abcam</td><td>ab14748</td><td>antibody to ATP synthase subunit ATP5A</td></tr><tr><td>anti-NDUFB6</td><td>Abcam</td><td>ab110244</td><td>antibody to Cx-1 subunit NDUFB6</td></tr><tr><td>anti-VDAC</td><td>Calbiochem</td><td>529534</td><td>antibody to VDAC</td></tr><tr><td>ECL HRP linked antibody</td><td>GE Health Care</td><td>NA931V</td><td>secondary antibody to ATP5A, NDUFB6 and VDAC</td></tr><tr><td>Blocking reagent</td><td>Biorad</td><td>170-6404</td><td></td></tr><tr><td>BSA</td><td></td><td></td><td></td></tr><tr><td>sodium chloride</td><td>Sigma</td><td>S9888</td><td></td></tr><tr><td>potassium chloride</td><td>Sigma</td><td>P9541</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>E3889</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>Silver staining Kit</td><td>Invitrogen</td><td>LC6070</td><td></td></tr></tbody></table>

analyzing supercomplexes of the mitochondrial electron transport chain with native electrophoresis, in-gel assays, and electroelution

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the cell, ATP. The ETC is composed of 5 massive protein complexes, which also assemble into supercomplexes called respirasomes (C-I, C-III, and C-IV) and synthasomes (C-V) that increase the efficiency of electron transport and ATP production. Various methods have been used for over 50 years to measure ETC function, but these protocols do not provide information on the assembly of individual complexes and supercomplexes. This protocol describes the technique of native gel polyacrylamide gel electrophoresis (PAGE), a method that was modified more than 20 years ago to study ETC complex structure. Native electrophoresis permits the separation of ETC complexes into their active forms, and these complexes can then be studied using immunoblotting, in-gel assays (IGA), and purification by electroelution. By combining the results of native gel PAGE with those of other mitochondrial assays, it is possible to obtain a completer picture of ETC activity, its dynamic assembly and disassembly, and how this regulates mitochondrial structure and function. This work will also discuss limitations of these techniques. In summary, the technique of native PAGE, followed by immunoblotting, IGA, and electroelution, presented below, is a powerful way to investigate the functionality and composition of mitochondrial ETC supercomplexes.

To visualize mitochondrial supercomplexes, freshly isolated mitochondria from mice were used17,18. Mitochondrial supercomplexes are sensitive to repeated cycles of freezing and thawing, leading to their disintegration, although this may be tolerable for some researchers. If freezing is necessary for storage, to ensure best results, samples should not undergo more than one cycle of freezing and thawing.
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Watch this Scientific Journal Video about Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution at JoVE.com

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

This protocol describes the separation of functional mitochondrial electron transport chain complexes (Cx) I-V and supercomplexes thereof using native electrophoresis to reveal information about their assembly and structure. The native gel can be subjected to immunoblotting, in-gel assays, and purification by electroelution to further characterize individual complexes.

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the ...

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Research

JoVE Journal

Biochemistry

14.0K Views.  University of Rochester. This protocol describes the separation of functional mitochondrial electron transport chain complexes (Cx) I-V and supercomplexes thereof using native electrophoresis to reveal information about their assembly and structure. The native gel can be subjected to immunoblotting, in-gel assays, and purification by electroelution to further characterize individual complexes. 

Video: Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

Biopanning bacterial display libraries is a proven technique for discovery of peptide affinity reagents, a robust alternative to antibodies. The semi-automated sorting method herein has streamlined biopanning to decrease the occurrence of false positives. Here we illustrate the thought process and techniques applied in evaluating candidates and minimizing downstream analysis.

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage by extracellular oxidants. This process of transporting electrons from intracellular reductants to extracellular oxidants is not well defined. Here we present spectrophotometric assays by C2C12 myotubes to monitor tPMET utilizing the extracellular electron acceptors: water-soluble tetrazolium salt-1 (WST-1) and 2,6-dichlorophenolindophenol (DPIP or DCIP). Through reduction of these electron acceptors, we are able to monitor this process in a real-time analysis. With the addition of enzymes such as ascorbate oxidase (AO) and superoxide dismutase (SOD) to the assays, we can determine which portion of tPMET is due to ascorbate export or superoxide production, respectively. While WST-1 was shown to produce stable results with low background, DPIP was able to be re-oxidized after the addition of AO and SOD, which was demonstrated with spectrophotometric analysis. This method demonstrates a real-time, multi-well, quick spectrophotometric assay with advantages over other methods used to monitor tPMET, such as ferricyanide (FeCN) and ferricytochrome c reduction.

The goal of this protocol is to spectrophotometrically monitor trans-plasma membrane electron transport utilizing extracellular electron acceptors and to analyze enzymatic interactions that may occur with these extracellular electron acceptors.

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage ...

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM c-Cyts) has recently emerged as a novel whole-cell analytical method to characterize the bacterial electron transport from the respiratory chain to the cell exterior, referred to as the extracellular electron transport (EET). While the pathway and kinetics of the electron flow during the EET reaction have been investigated, a whole-cell electrochemical method to examine the impact of cation transport associated with EET has not yet been established. In the present study, an example of a biochemical technique to examine the deuterium kinetic isotope effect (KIE) on EET through OM c-Cyts using a model microbe, Shewanella oneidensis MR-1, is described. The KIE on the EET process can be obtained if the EET through OM c-Cyts acts as the rate-limiting step in the microbial current production. To that end, before the addition of D2O, the supernatant solution was replaced with fresh media containing a sufficient amount of the electron donor to support the rate of upstream metabolic reactions, and to remove the planktonic cells from a uniform monolayer biofilm on the working electrode. Alternative methods to confirm the rate-limiting step in microbial current production as EET through OM c-Cyts are also described. Our technique of a whole-cell electrochemical assay for investigating proton transport kinetics can be applied to other electroactive microbial strains.

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Here we present a protocol of whole-cell electrochemical experiments to study the contribution of proton transport to the rate of extracellular electron transport via the outer-membrane cytochromes complex in Shewanella oneidensis MR-1.

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM ...

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

Biology

Hybrid Clear/Blue Native Electrophoresis for the Separation and Analysis of Mitochondrial Respiratory Chain Supercomplexes

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.

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 ...

Measuring Mitochondrial Electron Transfer Complexes in Previously Frozen Cardiac Tissue from the Offspring of Sow: A Model to Assess Exercise-Induced Mitochondrial Bioenergetics Changes

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis proposed and tested was that a regular maternal exercise of a sow during pregnancy would increase the mitochondrial efficiency of offspring heart bioenergetics. This hypothesis was tested by isolating mitochondria using a mild-isolation procedure to assess mitochondrial ETC and supercomplex profiles. The procedure described here allowed for the processing of previously frozen archived heart tissues and eliminated the necessity of fresh mitochondria preparation for the assessment of mitochondrial ETC complexes, supercomplexes, and ETC complex activity profiles. This protocol describes the optimal ETC protein complex measurement in multiplexed antibody-based immunoblotting and super complex assessment using blue-native gel electrophoresis.

Preparation of mitochondria-enriched samples from previously frozen archived solid tissues allowed the investigators to perform both functional and analytical assessments of mitochondria in various experimental modalities. This study demonstrates how to prepare mitochondria-enriched preparations from frozen heart tissue and perform analytical assessments of mitochondria.

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis ...

Mitochondrial Fractionation in Small Tissue Samples for Supercomplex Analysis

Isolation of Mitochondria for Mitochondrial Supercomplex Analysis from Small Tissue and Cell Culture Samples

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial electron transport chain organization, giving rise to the proposal of the "plasticity model." This model postulates the coexistence of different proportions of SCs and complexes depending on the tissue or the cellular metabolic status. The dynamic nature of the assembly in SCs would allow cells to optimize the use of available fuels and the efficiency of electron transfer, minimizing reactive oxygen species generation and favoring the ability of cells to adapt to environmental changes. 
More recently, abnormalities in SC assembly have been reported in different diseases such as neurodegenerative disorders (Alzheimer's and Parkinson's disease), Barth Syndrome, Leigh syndrome, or cancer. The role of SC assembly alterations in disease progression still needs to be confirmed. Nevertheless, the availability of enough amounts of samples to determine the SC assembly status is often a challenge. This happens with biopsy or tissue samples that are small or have to be divided for multiple analyses, with cell cultures that have slow growth or come from microfluidic devices, with some primary cultures or rare cells, or when the effect of particular costly treatments has to be analyzed (with nanoparticles, very expensive compounds, etc.). In these cases, an efficient and easy-to-apply method is required. This paper presents a method adapted to obtain enriched mitochondrial fractions from small amounts of cells or tissues to analyze the structure and function of mitochondrial SCs by native electrophoresis followed by in-gel activity assays or western blot.

Author Spotlight: Unveiling Oxidative Phosphorylation System Dynamics and Mitochondrial Roles in Health and Disease

This protocol describes a technique for the analysis of respiratory supercomplexes when only small amounts of samples are available.

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial ...

Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb the assembly and stability of OXPHOS complexes. This protocol describes the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE) in application to cultured human cells. First, mitochondria are extracted from the cells using digitonin, then using lauryl maltoside, the intact OXPHOS complexes are isolated from the mitochondrial membranes. The OXPHOS complexes are then resolved by gradient gel electrophoresis in the presence of the negatively charged dye, Coomassie blue, which prevents protein aggregation and ensures electrophoretic mobility of protein complexes towards the cathode. Finally, the OXPHOS complexes are detected by standard immunoblotting. Thus, BN-PAGE is a convenient and inexpensive technique that can be used to evaluate the assembly of entire OXPHOS complexes, in contrast to the basic SDS-PAGE allowing the study of only individual OXPHOS complex subunits.

This protocol demonstrates the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis. Here the method applied to cultured human cells is described.

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb ...

Blue Native Polyacrylamide Gel Electrophoresis: A Non-Denaturing Separation Technique for Analysis of Intact Mitochondrial Respiratory Chain Complexes

Source: Konovalova, S. <a href="https://www.jove.com/v/59269">Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting</a>. J. Vis. Exp. (2019)
In this video, we demonstrate an electrophoretic technique, called blue native PAGE, for the analysis of mitochondrial respiratory protein complexes in their natively folded and functionally active state. The anionic Coomassie blue dye causes the protein complexes to become negatively charged, allowing separation based on size and structure.

Source: Konovalova, S. Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis ...

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

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Chapters in this video

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Hybrid Clear/Blue Native Electrophoresis for the Separation and Analysis of Mitochondrial Respiratory Chain Supercomplexes

Measuring Mitochondrial Electron Transfer Complexes in Previously Frozen Cardiac Tissue from the Offspring of Sow: A Model to Assess Exercise-Induced Mitochondrial Bioenergetics Changes

Isolation of Mitochondria for Mitochondrial Supercomplex Analysis from Small Tissue and Cell Culture Samples

Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting

Blue Native Polyacrylamide Gel Electrophoresis: A Non-Denaturing Separation Technique for Analysis of Intact Mitochondrial Respiratory Chain Complexes