This work was supported by grant number &#34;PGC2018-095795-B-I00&#34; from Ministerio de Ciencia e Innovaci&#243;n (https://ciencia.sede.gob.es/) and by grants &#8220;Grupo de Referencia: E35_17R&#8221; and grant number &#8220;LMP220_21&#8221; from Diputaci&#243;n General de Arag&#243;n (DGA) (https://www.aragon.es/) to PF-S and RM-L.&#160;

NOTE: The composition of all culture media and buffers is specified in Table 1 and details related to all materials and reagents used in this protocol are listed in the Table of Materials.
1. Mitochondria isolation from cell culture
NOTE: The minimum volume of cells assayed has been ~30-50 &#181;L of packed cells (step 1.4). This can correspond approximately to at least two or three 100 mm cell culture plates or to one 150 mm plate at 80-90% of confluence, depending on the cell type (between 5 &#215; 106 and 107 cells in L929-derived cells or MDA-MB-468). Efficient cell breakage is a critical step.
<ol>
	<li>Grow adherent cells to approximately 80% confluence or suspended cells to an adequate density.</li>
	<li>Harvest cells by centrifugation at 500 &#215; g for 5 min (directly from the cell suspension in the case of non-adherent cells or after standard trypsinization with a trypsin-EDTA solution of 0.05% trypsin and 0.02% EDTA in 1x PBS, in the case of adherent cells).</li>
	<li>Wash cells 2x with cold 1x&#160;PBS and sediment them by centrifugation at 500 &#215; g for 5 min. 
	NOTE: From this point, all the steps must be performed at 4 &#176;C. Therefore, all the reagents must be cold and the tubes containing cells or mitochondria must be kept on ice.</li>
	<li>After the second centrifugation, discard the supernatant, estimate the cell pellet volume (cpv), and freeze the cells at -80 &#176;C for at least 15 min to facilitate membrane breaking in step 1.8. 
	NOTE: The protocol can be stopped at this point and the cells can be stored at -80 &#176;C for weeks. Usually, 10 or 15 mL graduated tubes with conical bottoms are appropriate.</li>
	<li>Let the cells thaw slowly on ice.</li>
	<li>Resuspend the packed cell pellet in a volume of hypotonic buffer equal to 7x the cpv (10 mM MOPS, 83 mM sucrose, pH 7.2) (e.g., 100 &#181;L of packed cells in 700 &#181;L of buffer). 
	NOTE: The volume must be enough to obtain efficient pops&#160;(see NOTE at step 1.8).</li>
	<li>Transfer the cell suspension into a Potter-Dounce homogenizer of the appropriate size and let the cells swell by incubating them in ice for 2 min. For example, for a volume of 700-800 &#181;L of cell suspension, a 1 mL capacity homogenizer is adequate.</li>
	<li>Break the cell membranes by performing eight to ten strokes in the homogenizer coupled to a motor-driven Teflon pestle rotating at 600 rpm. 
	NOTE: When making the strokes, it is important to create a vacuum that will increase the cell breakage efficiency along with the hypotonic and freezing actions for swelling and fragilizing the membranes, respectively. If it is well done, a &#34;pop&#34; sound will be heard when pulling down the homogenizer with a quick movement at each stroke.</li>
	<li>Add the same volume of hypertonic buffer to the cell suspension (7x the cpv) (30 mM MOPS, 250 mM sucrose, pH 7.2) to generate an isotonic environment.</li>
	<li>Transfer the homogenate into a 10-15 mL tube and centrifuge in a fixed rotor at 1,000 &#215; g for 5 min at 4 &#176;C. 
	NOTE: The original tube containing the unbroken cells can be used for this purpose.</li>
	<li>Transfer the supernatant, which contains the mitochondrial fraction, into 1.5 mL polypropylene tubes.</li>
	<li>OPTIONAL: To increase the yield of mitochondria, resuspend the pellet from step 1.10 in 7x the cell pellet volume of buffer A (10 mM Tris, 1 mM EDTA, 0.32 M sucrose, pH 7.4) by pipetting up and down and centrifuge at 1,000 &#215; g for 5 min at 4 &#176;C. Combine the new supernatant with the previous one before proceeding to step 1.13.</li>
	<li>Centrifuge in a microfuge at 16,000 &#215; g for 2 min at 4 &#176;C to collect the mitochondrial crude fraction.</li>
	<li>Discard the supernatant and resuspend each mitochondria-enriched pellet with 0.5 mL of buffer A combining the contents of two tubes into one and centrifuge under the same conditions as described in step 1.13. Repeat the same process until all the material is in only one tube.</li>
	<li>Discard the supernatant, resuspend the final pellet with 300 &#181;L of buffer A, and quantify mitochondrial protein concentration using the Bradford assay. Centrifuge again as before and proceed to section 3.</li>
</ol>
2. Isolation of mitochondria from small amounts of animal tissues
NOTE: The minimum amount of tissue to apply this protocol with a certain confidence depends on the cell type and its mitochondrial abundance but could be ~20-30 mg of tissue for most cases. The tissue samples can be fresh material or frozen samples. In the latter case, allow the samples to thaw in homogenization buffer placed on ice before starting the procedure.
<ol>
	<li>Weigh or estimate as closely as possible the amount (mg) of tissue.</li>
	<li>Cut the tissue with a pair of scissors and make 3-4 washes in homogenization buffer with the help of a strainer taking care to avoid losing the smaller pieces. 
	NOTE: This step is more important in tissues like skeletal muscle or heart than in the brain or softer tissues whose cells can be easily disaggregated directly by the homogenization step.</li>
	<li>Add fresh homogenization medium (4 mL per gram of liver, 10 mL per gram of heart, brown adipose tissue (BAT) or muscle, and 5 mL per gram of brain or kidney; see specific buffer composition for each case in Table 1).</li>
	<li>Transfer the pieces of tissue with buffer to the homogenizer and follow the optimal homogenization and mitochondria isolation protocol depending on the selected tissue.</li>
	<li>Isolation of mitochondria from liver, spleen, and kidney
	<ol>
		<li>Homogenize with four to six up-and-down strokes in the Elvehjem-Potter with a motor-driven Teflon pestle at 600 rpm.</li>
		<li>Transfer the homogenized tissue to a sterile centrifuge tube. Centrifuge in a swinging rotor at 1,000 &#215; g for 5 min at 4 &#176;C.</li>
		<li>Fill 1.5 mL polypropylene tubes with the supernatant obtained in the previous step. Centrifuge for 2 min at 16,000 &#215; g in a microfuge at 4 &#176;C and proceed hereafter as described before (steps 1.13 to 1.15)</li>
	</ol>
	</li>
	<li>Isolation of mitochondria from heart and muscle samples
	<ol>
		<li>Homogenize with six to eight strokes in the Elvehjem-Potter with a motor-driven Teflon pestle at 600 rpm. Transfer the homogenized tissue to a sterile centrifuge tube.</li>
		<li>Centrifuge in a swinging rotor at 1,000 &#215; g for 5 min at 4 &#176;C. Pour the supernatant into clean 1.5 mL polypropylene tubes. 
		NOTE: A second homogenization of the pellet obtained in step 2.6.2 can be performed with&#160;4-5 additional strokes and &#189; volume of homogenization buffer to increase the mitochondrial yield. The new supernatant (after centrifugation again at 1,000 &#215; g for 5 min at 4 &#176;C) can be combined with the previous one before proceeding to step 2.6.3. An alternative to increase the crude mitochondria yield is to dissociate heart and muscle tissue samples in trypsin solution before homogenization36,37. In this case, the trypsin has to be efficiently inactivated/removed before the mitochondrial membranes are solubilized with digitonin (step 3.2).</li>
		<li>Centrifuge at 16,000 &#215; g in a microfuge for 2 min at 4 &#176;C to obtain the crude mitochondrial fraction.</li>
		<li>Discard the supernatant and resuspend each mitochondria-enriched pellet with 0.5 mL of buffer AT combining the contents of two tubes into one and centrifuge under the same conditions as described in step 2.6.3. Repeat the same process until all the material is in only one tube.</li>
		<li>Discard the supernatant, resuspend the final pellet with 300 &#181;L of buffer A (no BSA), and quantify mitochondrial protein concentration using the Bradford assay. Centrifuge again as before and proceed to section 3.</li>
	</ol>
	</li>
	<li>Isolation of mitochondria from brain
	<ol>
		<li>Homogenize the pieces of the brain with 10-15 strokes using a Dounce-type glass homogenizer with a manually driven glass pestle. Transfer the homogenized tissue to a sterile centrifuge tube.</li>
		<li>Centrifuge in a swinging rotor at 1,000 &#215; g for 5 min at 4 &#176;C.</li>
		<li>Collect the supernatant into a clean centrifuge tube.</li>
		<li>Resuspend the pellet obtained in the previous centrifugation step in the same volume of medium AT used in the first homogenization. Re-homogenize the pellet by repeating the process described in step 2.7.1 using 5-10 passes and centrifuge the suspension in a swinging rotor at 1,000 &#215; g for 5 min at 4 &#176;C.</li>
		<li>Remove the supernatant and add it into the tube prepared in step 2.7.3. Centrifuge at 10,000 &#215; g in a fixed angle rotor for 10 min at 4 &#176;C to obtain the crude mitochondrial fraction.</li>
		<li>Discard the supernatant and resuspend the pellet into 1/2 the initial homogenization volume (step 2.7.1) of medium AT. Distribute the mitochondrial suspension into clean 1.5 mL polypropylene tubes and centrifuge at 16,000 &#215; g in a microfuge for 2 min at 4 &#176;C.</li>
		<li>Discard the supernatant and resuspend each mitochondria-enriched pellet with 0.5 mL of buffer AT combining the contents of two tubes into one and centrifuge under the same conditions as described in step 2.7.6. Repeat the same process until all the material is in only one tube.</li>
		<li>Discard the supernatant, resuspend the final pellet with 300 &#181;L of buffer A (no BSA), and quantify mitochondrial protein concentration using the Bradford assay. Centrifuge again as before and proceed to section 3.</li>
	</ol>
	</li>
	<li>Isolation of mitochondria from BAT
	<ol>
		<li>Homogenize with eight to ten up-and-down strokes in the Elvehjem-Potter with a motor-driven Teflon pestle at 600 rpm. Transfer the homogenized tissue to a sterile centrifuge tube.</li>
		<li>Centrifuge in a swinging rotor at 1,000 &#215; g for 5 min at 4 &#176;C.</li>
		<li>Fill 1.5 mL polypropylene tubes with the supernatant obtained in the previous step avoiding the upper fat layer. 
		&#8203;NOTE: A second homogenization of the pellet obtained in step 2.8.2 can be performed to increase the mitochondrial yield. The new supernatant (after centrifugation again at 1,000 &#215; g for 5 min at 4 &#176;C) can be combined with the previous one before proceeding to step 2.8.4.</li>
		<li>Centrifuge for 2 min at 16,000 &#215; g for 2 min in a microfuge at 4 &#176;C.</li>
		<li>Discard the supernatant and resuspend each mitochondria-enriched pellet with 0.5 mL of buffer AT2 combining the contents of two tubes into one and centrifuge under the same conditions as described in step 2.8.4. Repeat the same process until all the material is in only one tube.</li>
		<li>Discard the supernatant, resuspend the final pellet with 300 &#181;L of buffer A (no BSA), and quantify mitochondrial protein concentration using the Bradford assay. Centrifuge again as before and proceed to section 3.</li>
	</ol>
	</li>
</ol>
3. Preparation of samples for Blue Native analysis
<ol>
	<li>Resuspend the mitochondrial fractions (obtained from mammalian cell cultures or tissues) in BN sample buffer (50 mM NaCl, 50 mM Imidazole, 5 mM Aminocaproic acid, 4 mM PMSF) to obtain a protein concentration of around 10 mg/mL. See Table 2 for expected yields for the different types of samples.</li>
	<li>Solubilize mitochondrial membranes by adding digitonin (10% stock solution) to obtain a ratio of 4 g of digitonin/g of mitochondrial protein (4 &#181;L of digitonin 10% stock for 10 &#181;L of the mitochondrial suspension prepared in step 3.1). 
	NOTE: The detergent-to-protein ratio (g/g) must be optimized for each type of sample to obtain reproducible results; 2-8 g of digitonin/g of mitochondrial protein was found to be optimal for most tissues34,35.</li>
	<li>Mix by gently pipetting up and down. Incubate samples on ice for 5 min. 
	NOTE: After this step, the mitochondrial suspension should become clearer (shift from opaque to translucent); otherwise, it could indicate that the amount of detergent is insufficient for correct membrane solubilization.</li>
	<li>Centrifuge at full speed in a microfuge (20,000 &#215; g approx.) for 25 min at 4 &#176;C to remove insoluble material.</li>
	<li>Collect the supernatant in a fresh tube. Add to the supernatant a volume of 5% G-250 (Coomassie Blue G-250 5% in 0.75 M aminocaproic acid) equivalent to 1/3 of the initial resuspension volume (step 3.1, this would correspond to a final proportion of 1.6 g of Coomassie/g of protein) and mix by pipetting.</li>
	<li>Keep on ice before loading on the gel or freeze aliquots at -80 &#176;C.</li>
</ol>
4. Blue Native gel electrophoresis
NOTE: The electrophoresis is performed in the cold room (4-8 &#176;C). If there is no cold room facility, a cooling block can be introduced in the electrophoresis tank. Commercial 3-13% native polyacrylamide gels29 are used, but homemade gels of the desired gradient concentrations can also be used38,39.
<ol>
	<li>Load the upper and lower chambers with the cold cathode A and anode buffers, respectively. Alternatively, load the samples in the wells before carefully filling the upper chamber with cathode buffer A. 
	NOTE: Commercial electrophoresis buffers are used in our laboratory, but they can be prepared as indicated in Table 1.</li>
	<li>Load between 30 and 100 &#181;g of mitochondrial protein. 
	NOTE: Usually, 30-50 &#181;g of protein (for WB), or 40-100 &#181;g of protein (for IGA) are loaded per well in a 10 lane gel (0.5 x 0.15 cm wells). Cathode A buffer contains Coomassie Blue G-250 and has an intense blue color that makes it difficult to see the gel wells for sample loading. It is convenient to mark the center of each well, with a red marker, for example, to help in placing the pipette tip during this step. Native molecular weight markers can be useful when setting up the BN-PAGE technique to confirm that the gradient gels are properly formed and that the complexes and SCs pattern is the correct one.</li>
	<li>Run at 80-100 V for 25-30 min and then at 160-180 V, limiting the current to 12 mA/gel, until the dye reaches the bottom of the gel (~125-165 min in total).</li>
	<li>If in-gel activity assays (IGAs) are to be performed, substitute the cathode buffer A with cathode buffer B when the dye front is in the middle of the gel (~1 h after the beginning of the run). 
	NOTE: Although the gels can be documented without any staining just after the run since some bands are visible (mainly complex V, which can be considered an &#34;internal&#34; maker with a MW of ~600 kDa) due to their binding to G-250 dye, usually, SCs and individual complexes will not be visible without staining or IGA assays. The gels to be used in IGA assays or for WB should not be stained or fixed.</li>
	<li>Disassemble the gel from the cassette and continue with Coomassie blue staining (section 5), IGA analysis (section 6), or WB immunodetection (section 7).</li>
</ol>
5. Gel staining
<ol>
	<li>Stain the gel for 10-15 min at room temperature (RT) using Coomassie blue dye solutions (Coomassie dye R-250 at 0.25% in 40% methanol, 10% acetic acid; stain for 10-15 min).</li>
	<li>Destain with several washes (typically 4-5 x 15 min) in 40% methanol plus 10% acetic acid at RT.</li>
	<li>Document the gel.</li>
</ol>
6. In gel-activity (IGA) assays
<ol>
	<li>Prepare IGA solutions for the analysis of the different respiratory complexes before the end of the electrophoresis and maintain them in the dark (by using dark-colored plastic boxes or covering them with aluminum foil, for example). The composition of each buffer is detailed in Table 1.</li>
	<li>Place the gel or the lanes to be used in a plastic box as small as possible to accommodate it.</li>
	<li>Add enough volume of the appropriate solution to cover the gel (usually 5 or 10 mL for 5 or 10 gel lanes, respectively) and incubate at RT with gentle shaking (60-80 rpm) and away from light. 
	NOTE: The time of incubation depends on the complex to be analyzed and on the nature and amount of sample loaded; CI and CIV are the easiest and fastest to give results. Thus, CI activity will usually start to become visible after a few minutes, while CII and CIV need ~30 min to be observed and CV ~1.5-2 h. The reaction can continue for hours in all cases and the rate of signal intensification can be reduced by moving the incubation to a cold room (4-6 &#176;C), for example, for an overnight (ON) incubation. CIII activity works only for clear native electrophoresis (CN), not for BN34.</li>
	<li>When the appropriate bands have developed, stop the reactions by removing the assay solutions, washing 2x with distilled water, and fixing the gel with 40% methanol plus 10% acetic acid (except for CV which is fixed only with methanol) for 30 min.
	<ol>
		<li>To analyze CIV activity after CI detection by IGA, wash the gel 2x with distilled water, document CI activity, incubate the gel (without fixing it!) with 50 mM potassium phosphate buffer (pH 7.2) 2x for 30 min, and then with the complete CIV reaction buffer until the complex IV bands appear (usually by incubating ON at RT or 4 &#176;C).</li>
	</ol>
	</li>
	<li>Document the gels. 
	NOTE: For ATPase activity, as the developed bands are white, when documenting the gel, place it on a dark background so that the clear bands would be visible.</li>
</ol>
7. Western blot analysis
<ol>
	<li>Prepare transfer buffer according to Table 1 and keep at 4 &#176;C until the end of the electrophoresis.</li>
	<li>Place the gel in a tray and add transfer buffer. Incubate at RT for 10-15 min.</li>
	<li>Cut a piece of PVDF membrane of the same size as the gel and activate it in methanol for 10 s under agitation. Wash several times with distilled water and add transfer buffer. Incubate at RT for 10-15 min with gentle shaking.</li>
	<li>Prepare the transfer sandwich, from bottom to top, avoiding bubbles between the gel and the membrane, in the following order: black side of cassette-sponge-blotting paper-gel-membrane-blotting paper- sponge-clear side of cassette.</li>
	<li>Close and lock the cassette and put the sandwich in the transfer tank in the correct orientation for transfer (black side towards the negative pole).</li>
	<li>Fill the transfer tank with transfer buffer, add a magnetic stirrer to agitate the buffer, and connect the power supply at 80 V for 2 h or at 100 V for 1 h. Perform the transfer between 4 and 8 &#176;C (for example in the cold room) and with a cooling block in the transfer system.</li>
	<li>Retrieve the membrane and continue with a standard western blot protocol, using specific antibodies for the different respiratory complexes to be detected29.</li>
</ol>

Mitochondrial Fractionation in Small Tissue Samples for Supercomplex Analysis

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R., Flores-Garcia, M., Pedraza-Chaverri, J., Zazueta, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alteration+of+mitochondrial+supercomplexes+assembly+in+metabolic+diseases.">Alteration of mitochondrial supercomplexes assembly in metabolic diseases.</a> Biochim Biophys Acta Mol Basis Dis. 1866 (12), 165935 (2020).</li><li>Gonzalez-Rodriguez, P., 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=Disruption+of+mitochondrial+complex+I+induces+progressive+parkinsonism.">Disruption of mitochondrial complex I induces progressive parkinsonism.</a> Nature. 599 (7886), 650-656 (2021).</li><li>Novack, G. V., Galeano, P., Castano, E. M., Morelli, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+supercomplexes:+physiological+organization+and+dysregulation+in+age-related+neurodegenerative+disorders.">Mitochondrial supercomplexes: physiological organization and dysregulation in age-related neurodegenerative disorders.</a> Front Endocrinol (Lausanne). 11, 600 (2020).</li><li>Ramirez-Camacho, I., Flores-Herrera, O., Zazueta, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relevance+of+the+supramolecular+arrangements+of+the+respiratory+chain+complexes+in+human+diseases+and+aging.">The relevance of the supramolecular arrangements of the respiratory chain complexes in human diseases and aging.</a> Mitochondrion. 47, 266-272 (2019).</li><li>Hollinshead, K. E. R., 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=Respiratory+Supercomplexes+Promote+Mitochondrial+Efficiency+and+Growth+in+Severely+Hypoxic+Pancreatic+Cancer.">Respiratory Supercomplexes Promote Mitochondrial Efficiency and Growth in Severely Hypoxic Pancreatic Cancer.</a> Cell Rep. 33 (1), 108231 (2020).</li><li>Ikeda, 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=Mitochondrial+supercomplex+assembly+promotes+breast+and+endometrial+tumorigenesis+by+metabolic+alterations+and+enhanced+hypoxia+tolerance.">Mitochondrial supercomplex assembly promotes breast and endometrial tumorigenesis by metabolic alterations and enhanced hypoxia tolerance.</a> Nat Commun. 10 (1), 4108 (2019).</li><li>Kamada, S., Takeiwa, T., Ikeda, K., Horie, K., Inoue, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+roles+of+COX7RP+and+mitochondrial+oxidative+phosphorylation+in+breast+cancer.">Emerging roles of COX7RP and mitochondrial oxidative phosphorylation in breast cancer.</a> Front Cell Dev Biol. 10, 717881 (2022).</li><li>Marco-Brualla, J., 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=Mutations+in+the+ND2+subunit+of+mitochondrial+complex+I+are+sufficient+to+confer+increased+tumorigenic+and+metastatic+potential+to+cancer+cells.">Mutations in the ND2 subunit of mitochondrial complex I are sufficient to confer increased tumorigenic and metastatic potential to cancer cells.</a> Cancers (Basel). 11 (7), 1027 (2019).</li><li>Moreno-Loshuertos, R., 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=How+hot+can+mitochondria+be?+Incubation+at+temperatures+above+43+degrees+C+induces+the+degradation+of+respiratory+complexes+and+supercomplexes+in+intact+cells+and+isolated+mitochondria.">How hot can mitochondria be? Incubation at temperatures above 43 degrees C induces the degradation of respiratory complexes and supercomplexes in intact cells and isolated mitochondria.</a> Mitochondrion. 69, 83-94 (2023).</li><li>Vonck, J., Schafer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supramolecular+organization+of+protein+complexes+in+the+mitochondrial+inner+membrane.">Supramolecular organization of protein complexes in the mitochondrial inner membrane.</a> Biochim Biophys Acta. 1793 (1), 117-124 (2009).</li><li>Althoff, T., Mills, D. J., Popot, J. L., 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=Arrangement+of+electron+transport+chain+components+in+bovine+mitochondrial+supercomplex+I1III2IV1.">Arrangement of electron transport chain components in bovine mitochondrial supercomplex I1III2IV1.</a> EMBO J. 30 (22), 4652-4664 (2011).</li><li>Cogliati, S., 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=Mechanism+of+super-assembly+of+respiratory+complexes+III+and+IV.">Mechanism of super-assembly of respiratory complexes III and IV.</a> Nature. 539 (7630), 579-582 (2016).</li><li>Gonzalez-Franquesa, A., 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=Mass-spectrometry-based+proteomics+reveals+mitochondrial+supercomplexome+plasticity.">Mass-spectrometry-based proteomics reveals mitochondrial supercomplexome plasticity.</a> Cell Rep. 35 (8), 109180 (2021).</li><li>Wittig, I., 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=Features+and+applications+of+blue-native+and+clear-native+electrophoresis.">Features and applications of blue-native and clear-native electrophoresis.</a> Proteomics. 8 (19), 3974-3990 (2008).</li><li>Wittig, I., 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=Native+electrophoretic+techniques+to+identify+protein-protein+interactions.">Native electrophoretic techniques to identify protein-protein interactions.</a> Proteomics. 9 (23), 5214-5223 (2009).</li><li>Garcia-Cazarin, M. L., Snider, N. N., Andrade, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+isolation+from+skeletal+muscle.">Mitochondrial isolation from skeletal muscle.</a> J Vis Exp. (49), e2452 (2011).</li><li>Lai, N., 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=Isolation+of+mitochondrial+subpopulations+from+skeletal+muscle:+Optimizing+recovery+and+preserving+integrity.">Isolation of mitochondrial subpopulations from skeletal muscle: Optimizing recovery and preserving integrity.</a> Acta Physiol (Oxf). 225 (2), e13182 (2019).</li><li>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=Native+electrophoresis+for+isolation+of+mitochondrial+oxidative+phosphorylation+protein+complexes.">Native electrophoresis for isolation of mitochondrial oxidative phosphorylation protein complexes.</a> Methods Enzymol. 260, 190-202 (1995).</li><li>Wittig, I., Braun, H. P., 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=Blue+native+PAGE.">Blue native PAGE.</a> Nat Protoc. 1 (1), 418-428 (2006).</li><li>Chomyn, A., 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=Platelet-mediated+transformation+of+mtDNA-less+human+cells:+analysis+of+phenotypic+variability+among+clones+from+normal+individuals--and+complementation+behavior+of+the+tRNALys+mutation+causing+myoclonic+epilepsy+and+ragged+red+fibers.">Platelet-mediated transformation of mtDNA-less human cells: analysis of phenotypic variability among clones from normal individuals--and complementation behavior of the tRNALys mutation causing myoclonic epilepsy and ragged red fibers.</a> Am J Hum Genet. 54 (6), 966-974 (1994).</li><li>Moreno-Loshuertos, R., 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=Differences+in+reactive+oxygen+species+production+explain+the+phenotypes+associated+with+common+mouse+mitochondrial+DNA+variants.">Differences in reactive oxygen species production explain the phenotypes associated with common mouse mitochondrial DNA variants.</a> Nat Genet. 38 (11), 1261-1268 (2006).</li><li>Fern&#225;ndez-Vizarra, E., Fern&#225;ndez-Silva, P., Enr&#237;quez, J. A., Celis, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Biology (Third Edition). , 69-77 (2006).</li><li>Cogliati, S., Herranz, F., Ruiz-Cabello, J., Enr&#237;quez, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digitonin+concentration+is+determinant+for+mitochondrial+supercomplexes+analysis+by+BlueNative+page.">Digitonin concentration is determinant for mitochondrial supercomplexes analysis by BlueNative page.</a> Biochim Biophys Acta Bioenerg. 1862 (1), 148332 (2021).</li></ol>

The authors declare no conflicts of interest.

The methodological adaptations introduced in the protocols described&#160;here are intended to avoid losses and increase the yield while maintaining mitochondrial complex activities (which is crucial when the availability of enough amounts of samples is compromised) and reproduce the tissue&#39;s or cell line&#39;s expected pattern of SCs (see Figure 2C). With this purpose and since a high mitochondrial purity is not required to properly detect the SCs, the number of steps, times, and volumes have been reduced whenever this was possible.
Thus, after homogenization and removal of nuclei and unbroken cells by low-speed centrifugation, the supernatants containing the mitochondria are transferred to 1.5 mL polypropylene tubes, and the subsequent centrifugations to obtain the crude mitochondrial fractions are performed in a microfuge by increasing the speed and reducing the times and volumes with respect to conventional protocols. This allows the processing of several samples in a relatively short time: a close estimation is that 10 samples can be processed in ~2-2.5 h (from the start of the homogenization (protocol step 1.6. or equivalent for tissues) to the obtention of the SCs fraction ready to be loaded in a gel (protocol step 3.7)&#160;and minimizes the losses while keeping activities. Another change with respect to the original Sch&#228;gger&#39;s group protocols is to substitute, after membrane solubilization for the obtention of the SCs fraction, an ultracentrifugation step by a simple microfuge centrifugation (protocol step 3.4).
There are some critical steps in the procedure. First, the efficiency of cell breakage, especially in the case of cultured cells (see note after protocol step 1.8) and particularly in the case of small cells. Tissues and cell lines present a variable difficulty degree in cell breakage: for example, in the heart and muscle, this step is usually less efficient than in the liver or spleen, but it is compensated in yield because the former have a higher mitochondrial content. One alternative to improve mitochondrial yield when isolated from muscle or heart would be the treatment of samples with trypsin before homogenization. The brain is another tissue that presents low efficiency in cell breakage because of the manual homogenization, which is milder and less efficient and probably also because of the higher variation in cell types in this tissue, as it has been reported previously 42. This can be partially compensated by a second homogenization step as suggested in our protocol (step 2.7.4). On the other hand, if cell breakage is excessive, the final sample containing SCs could be contaminated with nuclear DNA fragments that may interfere with the electrophoretic running. This problem is shown as an increase in the viscosity of the sample when digitonin is added to solubilize the mitochondrial membranes and can be solved by treatment with DNAse I.
Second, if the detergent-to-protein ratio is too low, that would result in less efficient membrane solubilization and the possibility of aggregate formation. This could show in the gels as poor resolution in high molecular weight SCs (see Figure 4) or as CIV giving multiple bands corresponding to oligomers43. If the ratio is too high, it could lead to disaggregation of SCs (mainly CI-SCs). Third, it is important to avoid heating samples during the whole procedure (see Figure 4) and avoid repeated cycles of freezing-thawing since they may affect SC assembly status.
In Table 2, a summary of expected yields is presented for several cell types and tissue samples. The values are approximate and will depend on the cell breakage efficiency and the nature of the source. In the case of tissues, if the starting material is frozen it tends to give a higher yield than with fresh samples. An estimation of the number of lanes (considering a load between 50 and 60 &#181;g of mitochondrial protein per lane) that can be loaded is presented.
The protocols presented here cover a variety of tissues and cell types with small variations, they can be used with fresh or frozen samples and require simple equipment and a relatively low cost, not involving the use of expensive purification kits or gradients. However, they may need some adaptations to be used with particular cell types or samples, as well as when specific mitochondrial populations, such as brain synaptic mitochondria or muscle subsarcolemmal mitochondria, for example, are the object of analysis.
Since the precise physiological roles as well as the potential involvement of SCs in pathological situations remains to be defined, the analysis of their dynamics and the factors affecting their assembly, stability, and function is needed. Our protocols can represent valuable tools that allow&#160;to obtain reproducible results on the SCs&#160;patterns with small sample sizes and from a variety of cell types and tissues, particularly when multiple experimental conditions need to be compared29.

Supercomplexes (SCs) are supramolecular associations between individual respiratory chain complexes1,2. Since the initial identification of SCs and the description of their composition by the group of Sch&#228;gger2,3, later confirmed by other groups, it was established that they contain respiratory complexes I, III, and IV (CI, CIII, and CIV, respectively) in different stoichiometries. Two main populations of SCs can be defined, those containing CI (and either CIII alone or CIII and CIV) and with very high molecular weight (MW, starting ~1.5 MDa for the smaller SC: CI + CIII2) and those containing CIII and CIV but not CI, with much smaller size (such as CIII2 + CIV with ~680 kDa). These SCs coexist in the inner mitochondrial membrane with free complexes, also in different proportions. Thus, while CI is mostly found in its associated forms (that is, in SCs: ~80% in bovine heart and more than 90% in many human cell types)3, CIV is very abundant in its free form (more than 80% in bovine heart), with CIII showing a more balanced distribution (~40% in its more abundant free form, as a dimer, in bovine heart).
While their existence is now generally accepted, their precise role is still under debate4,5,6,7,8,9,10. According to the plasticity model, different proportions of SCs and individual complexes can exist depending on the cell type or the metabolic status1,7,11. This dynamic nature of the assembly would allow cells to regulate the use of available fuels and the efficiency of the oxidative phosphorylation (OXPHOS) system in response to environmental changes4,5,7. SCs could also contribute to control the reactive oxygen species generation rate and participate in the stabilization and turnover of individual complexes4,12,13,14. Modifications of the SC assembly status have been described in association with different physiological and pathological situations15,16 and with the aging process17.
Thus, changes in the SC patterns have been described in yeast depending on the carbon source used for growth2&#160;and in cultured mammalian cells when glucose is substituted by galactose4. Modifications have also been reported in mouse liver after fasting8 and in astrocytes when mitochondrial fatty acid oxidation is blocked18. In addition, a decrease or alterations in SCs and OXPHOS have been found in Barth syndrome19, heart failure20, several metabolic21 and neurological22,23,24 disorders, and different tumors25,26,27,28. Whether these alterations in SC assembly and levels are a primary cause or represent secondary effects in these pathological situations is still under investigation15,16. Different methodologies can give information about the assembly and function of SCs; these include activity measurements8,29, ultrastructural analysis30,31, and proteomics32,33. A useful alternative that is increasingly being employed and is the starting point for some of the previously mentioned methodologies is the direct determination of SC assembly status by Blue native (BN) electrophoresis developed for this purpose by Sch&#228;gger&#39;s group34,35.
This approach requires reproducible and efficient procedures to obtain and solubilize mitochondrial membranes and can be complemented by other techniques such as in-gel activity analysis (IGA), second-dimension electrophoresis, and western blot (WB). A limitation in the studies on SC dynamics by BN electrophoresis can be the amount of starting cells or tissue samples. We present a series of protocols for the analysis of SC assembly and function, adapted from Sch&#228;gger&#39;s group methods, that can be applied to fresh or frozen cell or tissue samples starting from as little as 20 mg of tissue.

<table><tbody><tr><td>Acetic acid</td><td>PanReac</td><td>131008</td><td></td></tr><tr><td>Aminocaproic acid</td><td>Fluka Analytical</td><td>7260</td><td></td></tr><tr><td>ATP</td><td>Sigma-Aldrich</td><td>A2383</td><td></td></tr><tr><td>Bis Tris</td><td>Acrons Organics</td><td>327721000</td><td></td></tr><tr><td>Bradford assay</td><td>Biorad</td><td>5000002</td><td></td></tr><tr><td>Coomassie Blue G-250</td><td>Serva</td><td>17524</td><td></td></tr><tr><td>Coomassie Blue R-250</td><td>Merck</td><td>1125530025</td><td></td></tr><tr><td>Cytochrome c</td><td>Sigma-Aldrich</td><td>C2506</td><td></td></tr><tr><td>Diamino&amp;nbsp; benzidine (DAB)</td><td>Sigma-Aldrich</td><td>D5637</td><td></td></tr><tr><td>Digitonin</td><td>Sigma-Aldrich</td><td>D5628</td><td></td></tr><tr><td>EDTA</td><td>PanReac</td><td>131669</td><td></td></tr><tr><td>EGTA</td><td>Sigma-Aldrich</td><td>E3889</td><td></td></tr><tr><td>Fatty acids free BSA</td><td>Roche</td><td>10775835001</td><td></td></tr><tr><td>Glycine</td><td>PanReac</td><td>A1067</td><td></td></tr><tr><td>Homogenizer Teflon pestle</td><td>Deltalab</td><td>196102</td><td></td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>I2399</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>PanReac</td><td>121512</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>PanReac</td><td>121509</td><td></td></tr><tr><td>Mannitol</td><td>Sigma-Aldrich</td><td>M4125</td><td></td></tr><tr><td>Methanol</td><td>Labkem</td><td>MTOL-P0P</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>PanReac</td><td>131404</td><td></td></tr><tr><td>Mini Trans-Blot Cell</td><td>BioRad</td><td>1703930</td><td></td></tr><tr><td>MOPS</td><td>Sigma-Aldrich</td><td>M1254</td><td></td></tr><tr><td>MTCO1 Monoclonal Antibody</td><td>Invitrogen</td><td>459600</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S9888</td><td></td></tr><tr><td>NADH</td><td>Roche</td><td>10107735001</td><td></td></tr><tr><td>NativePAGE 3 to 12% Mini Protein Gels</td><td>Invitrogen</td><td>BN1001BOX</td><td></td></tr><tr><td>NativePAGE Cathode Buffer Additive (20x)</td><td>Invitrogen</td><td>BN2002</td><td></td></tr><tr><td>NativePAGE Running Buffer (20x)&amp;nbsp;</td><td>Invitrogen</td><td>BN2001</td><td></td></tr><tr><td>NDUFA9 Monoclonal Antibody</td><td>Invitrogen</td><td>459100</td><td></td></tr><tr><td>Nitroblue tetrazolium salt (NBT)</td><td>Sigma-Aldrich</td><td>N6876</td><td></td></tr><tr><td>Pb(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>228621</td><td></td></tr><tr><td>PDVF Membrane</td><td>Amersham</td><td>10600023</td><td></td></tr><tr><td>Phenazine methasulfate (PMS)</td><td>Sigma-Aldrich</td><td>P9625</td><td></td></tr><tr><td>Pierce ECL Substrate</td><td>Thermo Scientific</td><td>32106</td><td></td></tr><tr><td>PMSF</td><td>Merck</td><td>PMSF-RO</td><td></td></tr><tr><td>SDHA Monoclonal Antibody</td><td>Invitrogen</td><td>459200</td><td></td></tr><tr><td>Sodium succinate</td><td>Sigma-Aldrich</td><td>S2378</td><td></td></tr><tr><td>Streptomycin/penicillin</td><td>PAN biotech</td><td>P06-07100</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S3089</td><td></td></tr><tr><td>Tris</td><td>PanReac</td><td>A2264</td><td></td></tr><tr><td>UQCRC1 Monoclonal Antibody</td><td>Invitrogen</td><td>459140</td><td></td></tr><tr><td>XCell&amp;nbsp;SureLock Mini-Cell</td><td>Invitrogen</td><td>&amp;nbsp;EI0001</td><td></td></tr></tbody></table>

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.

The yields of mitochondria obtained following the above-described protocols vary depending on several factors such as the cell line or tissue type, the nature of the samples (i.e., if fresh or frozen tissues are used), or the efficiency of the homogenization process. Expected yields of mitochondria from different cell lines and tissues are collected in Table 2. Once the mitochondrial fractions have been obtained, the next step is the analysis of respiratory SCs pattern, which is performed after the crude mitochondrial sample solubilization and electrophoretic separation by BN-PAGE followed by IGA-analysis or WB immunodetection. Figure 1 shows the aspect of unstained gel lanes just after the run and the pattern of bands after standard Coomassie staining of a cultured cell line and a mouse liver mitochondrial sample.
In Figure 2A,B, clear differences between the SC assembly patterns in human and mouse cells after using IGA assays can be observed. Thus, free complex I is observed in mouse cells whereas it is not detectable in human mitochondria. Complex IV-IGA pattern is very similar in both human cell lines (Figure 2B) while presenting differences between the two mouse cell lines analyzed in Figure 2A. These differences are due to the fact that BL6 cells express a mutant variant of SCAF18.
The SC patterns, obtained with the protocol for mitochondrial isolation described here to work with small samples, are maintained with respect to the &#34;conventional&#34; protocols used for larger samples. This can be seen in Figure 2C, by comparing lanes 1 and 6 (obtained after mitochondrial isolation using a conventional protocol from 3 g of liver and 1.1 g of BAT) with, respectively, lanes 2 and 5 (obtained using the protocols here presented from around 0.1 g of both tissues). As proposed in the plasticity model, the relative proportion of respiratory complexes and SCs varies depending on the cell type and the metabolic state. As shown in Figure 2C,D, the different tissues show different SC assembly patterns. Thus, brain mitochondria show very low levels of free complex I and a higher proportion of SCs when compared with the other tissues, and BAT is characterized by its low levels of CV (Figure 2A) and high amounts of SC III + IV (Figure 2D). Heart mitochondria present high levels of SCs and CV. CV assembly patterns are very similar between the cultured cell line and a mouse liver mitochondrial sample (Figure 2E).
SC assembly can also be analyzed by WB as shown in Figure 3 for two samples obtained from spleen control and tumor cells. In this case, 30-50 &#181;g of mitochondrial protein are usually enough to detect the different complexes and SCs with the specific antibodies. Figure 4 shows some of the main pitfalls that can happen during the application of these protocols. Wrong gel gradients can produce abnormal and undesired migration patterns as in Figure 4A, lane 1, where the SCs remain close to the well in the upper part of the lane due to a higher-than-normal acrylamide concentration in the gel. Incubation of the samples, even for relatively short times, at high temperatures (here 37 &#176;C and 40 &#176;C for 10 min) causes degradation of SCs and CI (Figure 4A lanes 2-4). Likewise, the detergent-to-protein ratio is critical for good results and adequate gel resolution. Below a ratio of 2 g of digitonin/g of protein, the band resolution is progressively reduced and at the lower concentration (0.5 g/g) only a smear in the area corresponding to SCs is visible (Figure 4B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66771/66771fig01.jpg" /> 
Figure 1: Pattern of mitochondrial complexes and supercomplexes after BNGE separation of digitonin-permeabilized mitochondria from a culture cell line (L929Balbc) and mouse liver. The lanes on the left (1 and 2) represent the aspect of the unstained gel just after the run, where only CV is detectable, while the lanes on the right (3 and 4) show the pattern of bands after Coomassie staining. Abbreviations: BNGE = Blue Native gel electrophoresis; CV = complex V; SCs = supercomplexes. <a href="https://www.jove.com/files/ftp_upload/66771/66771fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66771/66771fig02.jpg" /> 
Figure 2: IGA-analysis of mitochondria isolated from different cell lines and mouse tissues. (A,B) In-gel activity of the indicated complexes in mitochondria isolated from (A) mouse or (B) human cell lines. L929Balbc is a transmitochondrial cell line generated in our laboratory by transferring mitochondria from Balb/cJ mouse platelets to &#961;&#176;L929neo cells as described previously40,41. BL6 fibroblasts are an immortalized cell line generated in our lab from an ear biopsy of a BL6 mouse. (C) CI-IGA patterns from mouse mitochondria isolated using either a conventional method starting with 3 g of liver (lane 1) and with 1.1 g of BAT (lane 6) or the protocol presented here and starting with around 0.1 g of tissue in all cases (lane 2, liver; lane 3, heart; lane 4, brain, and lane 5 BAT). (D) CIV-IGA patterns (performed after CI-IGA shown in panel 2C, lanes 2-5) in mouse liver, heart, brain, and BAT mitochondria. (E) CV-IGA activity analyzed in mouse cultured cells and liver. Around 60-75 &#181;g of mitochondrial protein per lane were loaded. Abbreviations: IGA = in-gel activity; SCs = supercomplexes; CI = complex I; BAT = brown adipose tissue. <a href="https://www.jove.com/files/ftp_upload/66771/66771fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66771/66771fig03.jpg" /> 
Figure 3: WB immunodetection of the different mitochondrial complexes and supercomplexes in spleen samples. After Blue Native PAGE separation of mitochondrial complexes and SCs obtained from spleen control (Spleen M9) and tumor (Tumor M9) samples, they were transferred to a PVDF membrane and sequentially hybridized with antibodies that recognize the indicated CI, CIII, CIV, and CII subunits (panels A-D, respectively). Around 50 &#181;g of mitochondrial protein were loaded per lane. Asterisks indicate the signal remaining from a previous western blot. Abbreviations: SC = supercomplex; CI = complex I; CIII = complex III; CIV = complex IV; CII = complex II. <a href="https://www.jove.com/files/ftp_upload/66771/66771fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66771/66771fig04v2.jpg" /> 
Figure 4: Pitfalls in SC analysis by Blue-Native PAGE. CI IGA-analysis of mitochondria isolated from (A) fresh mouse liver and (B) frozen rat liver under different solubilization conditions. (A) Lane 1, wrong gel gradient concentration (8-13% instead of the normal 3-13%). Lanes 2-4, samples incubated for 10 min on ice or at 37 &#176;C and 40 &#176;C before loading, respectively. (B) Pattern after IGA for CI of mitochondria obtained from frozen rat liver using different digitonin/protein ratios for solubilization. Abbreviations: SC = supercomplex; PAGE = polyacrylamide gel electrophoresis; CI = complex I; IGA = in-gel activity. <a href="https://www.jove.com/files/ftp_upload/66771/66771fig04largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Medium</td>
			<td colspan="3">Composition</td>
		</tr>
		<tr>
			<td>Hypotonic buffer</td>
			<td colspan="3">10 mM MOPS, 83 mM sucrose, pH 7.2</td>
		</tr>
		<tr>
			<td>Hypertonic buffer</td>
			<td colspan="3">30 mM MOPS, 250 mM sucrose, pH 7.2</td>
		</tr>
		<tr>
			<td>Homogenization Buffer A</td>
			<td colspan="3">10 mM Tris, 1 mM EDTA, 0.32 M sucrose, pH 7.4</td>
		</tr>
		<tr>
			<td>Homogenization Buffer AT</td>
			<td colspan="3">225 mM mannitol, 1 mM EGTA, 75 mM sucrose, 0.01% BSA pH 7.4</td>
		</tr>
		<tr>
			<td>Homogenization Buffer AT2</td>
			<td colspan="3">225 mM mannitol, 1 mM EGTA, 75 mM sucrose, 0.02% BSA pH 7.4</td>
		</tr>
		<tr>
			<td>BN sample buffer</td>
			<td colspan="3">50 mM NaCl, 50 mM Imidazole, 2 mM Aminocaproic acid, 1 mM EDTA pH 7.0</td>
		</tr>
		<tr>
			<td>Digitonin solution</td>
			<td colspan="3">10% digitonin in 50 mM NaCl, 50 mM Imidazole, 5 mM Aminocaproic, 4 mM PMSF</td>
		</tr>
		<tr>
			<td>BN loading buffer</td>
			<td colspan="3">5% Coomassie Blue G in 0.75 M aminocaproic acid</td>
		</tr>
		<tr>
			<td>BN cathode buffer A</td>
			<td colspan="3">50 mM tricine, 15 mM Bis-Tris pH =7.0 (4 &#176;C), 0.02% G-250 (Coomassie brilliant blue G-250)</td>
		</tr>
		<tr>
			<td>BN cathode buffer B</td>
			<td colspan="3">50 mM tricine, 15 mM Bis-Tris pH=7.0 (4 &#176;C), 0.002% G-250</td>
		</tr>
		<tr>
			<td>BN anode buffer</td>
			<td colspan="3">50 mM Bis-Tris, pH=7.0</td>
		</tr>
		<tr>
			<td>Complex I IGA substrate</td>
			<td colspan="3">5 mM Tris-HCl pH 7.4, 0.1 mg/mL NADH, 2.5 mg/mL Nitroblue Tetrazolium (NBT)</td>
		</tr>
		<tr>
			<td>Complex II IGA substrate</td>
			<td colspan="3">50 mM Potassium phosphate buffer pH 7.4, 20 mM sodium succinate, 0.2 mM phenazine methosulfate (PMS), 2.5 mg/mL NBT</td>
		</tr>
		<tr>
			<td>Complex III IGA substrate</td>
			<td colspan="3">50 mM Potassium phosphate buffer pH 7.2, 0.05% diaminobenzidine (DAB)</td>
		</tr>
		<tr>
			<td>Complex IV IGA substrate</td>
			<td colspan="3">Same solution as CIII plus 50 &#956;M cytochrome c</td>
		</tr>
		<tr>
			<td>Complex V IGA substrate</td>
			<td colspan="3">35 mM Tris, 270 mM glycine pH 8.3, 14 mM MgSO4, 0.2% Pb(NO3)2, 8 mM ATP&#160;</td>
		</tr>
		<tr>
			<td>Staining solution</td>
			<td colspan="3">Coomassie dye R-250 at 0.25% in 40% methanol and 10% Acetic acid</td>
		</tr>
		<tr>
			<td>Destaining/fixing solution</td>
			<td colspan="3">40% Methanol, 10% Acetic Acid</td>
		</tr>
		<tr>
			<td>Transfer buffer</td>
			<td colspan="3">48 mM Tris, 39 mM Glycine, 20% methanol</td>
		</tr>
	</tbody>
</table>
Table 1: Buffers and media composition.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell type/tissue</td>
			<td>Starting amount</td>
			<td>Expected yield (&#181;g mitochondrial protein)</td>
			<td>Number of lanes (50-60 &#181;g/lane)</td>
		</tr>
		<tr>
			<td>Cultured cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MDA-MB-468</td>
			<td>100 &#181;L vpc</td>
			<td>300-400 &#181;g</td>
			<td>6-9</td>
		</tr>
		<tr>
			<td>143B-derived&#160;</td>
			<td>100 &#181;L vpc</td>
			<td>200-350 &#181;g</td>
			<td>4-7</td>
		</tr>
		<tr>
			<td>Mouse Fibroblasts</td>
			<td>100 &#181;L vpc</td>
			<td>150-200 &#181;g</td>
			<td>3-5</td>
		</tr>
		<tr>
			<td>Heart</td>
			<td>100 mg</td>
			<td>400-500 &#181;g</td>
			<td>10-12*</td>
		</tr>
		<tr>
			<td>Skeletal muscle</td>
			<td>100 mg</td>
			<td>300-400 &#181;g</td>
			<td>8-10*</td>
		</tr>
		<tr>
			<td>Liver</td>
			<td>100 mg</td>
			<td>400-500 &#181;g</td>
			<td>8-10</td>
		</tr>
		<tr>
			<td>Spleen</td>
			<td>50 mg</td>
			<td>150-250 &#181;g</td>
			<td>3-5</td>
		</tr>
		<tr>
			<td>BAT</td>
			<td>100 mg</td>
			<td>200-300 &#181;g</td>
			<td>4-6</td>
		</tr>
		<tr>
			<td>Brain</td>
			<td>100 mg</td>
			<td>250-300 &#181;g</td>
			<td>5-8</td>
		</tr>
	</tbody>
</table>
Table 2:&#160;Expected yields of mitochondria from different cell types and mouse tissues. The mitochondrial fractions obtained from heart or skeletal muscle are purer than those obtained from other tissues. Thus, smaller amounts can be loaded in the gels (*).

Read this Scientific Article Protocol about Isolation of Mitochondria for Mitochondrial Supercomplex Analysis from Small Tissue and Cell Culture Samples at JoVE.com

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.

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 ...

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Nanyang Technological University

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Biochemistry

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

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

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.

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 ...

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

Rapid Isolation of the Mitoribosome from HEK Cells

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative phosphorylation complexes encoded by the mitochondrial genome. Since all proteins synthesized by human mitoribosomes are integral membrane proteins, human mitoribosomes are tethered to the mitochondrial inner membrane during translation. Compared to the cytosolic ribosome the mitoribosome has a sedimentation coefficient of 55S, half the rRNA content, no 5S rRNA and 36 additional proteins. Therefore, a higher protein-to-RNA ratio and an atypical structure make the human mitoribosome substantially distinct from its cytosolic counterpart.
Despite the central importance of the mitoribosome to life, no protocols were available to purify the intact complex from human cell lines. Traditionally, mitoribosomes were isolated from mitochondria-rich animal tissues that required kilograms of starting material. We reasoned that mitochondria in dividing HEK293-derived human cells grown in nutrient-rich expression medium would have an active mitochondrial translation, and, therefore, could be a suitable source of material for the structural and biochemical studies of the mitoribosome. To investigate its structure, we developed a protocol for large-scale purification of intact mitoribosomes from HEK cells. Herein, we introduce nitrogen cavitation method as a faster, less labor-intensive and more efficient alternative to traditional mechanical shear-based methods for cell lysis. This resulted in preparations of the mitoribosome that allowed for its structural determination to high resolution, revealing the composition of the intact human mitoribosome and its assembly intermediates. Here, we follow up on this work and present an optimized and more cost-effective method requiring only ~1010 cultured HEK cells. The method can be employed to purify human mitoribosomal translating complexes, mutants, quality control assemblies and mitoribosomal subunits intermediates. The purification can be linearly scaled up tenfold if needed, and also applied to other types of cells.

Mitochondria have specialized ribosomes that diverged from their bacterial and cytoplasmic counterparts. Here we show how mitoribosomes can be obtained from their native compartment in HEK cells. The method involves isolation of mitochondria from suspension cells and consequent purification of mitoribosomes.

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Medicine

Stable Isotope Tracing & LC-MS Analysis to Measure DHODH and SDH Activities

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

Author Spotlight: Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals  

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

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Mitochondrial Isolation from Rodents for High-Quality Bioenergetic Studies

Robust Mitochondrial Isolation from Rodent Cardiac Tissue

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry to study metabolic impairments and disease. Consistent mitochondrial quality is necessary to properly investigate mitochondrial physiology and bioenergetics; however, many different published isolation methods are available for researchers. Although different experimental strategies require different isolation methods, the basic principles and procedures are similar. This protocol details a method capable of extracting well-coupled mitochondria from a variety of tissue sources, including small animals and cells. The steps outlined include organ dissection, mitochondrial purification, protein quantification, and various quality control checks. The primary quality control metric used to identify high-quality mitochondria is the respiratory control ratio (RCR). The RCR is the ratio of the respiratory rate during oxidative phosphorylation to the rate in the absence of ADP. Alternative metrics are discussed. While high RCR values relative to their tissue source are obtained using this protocol, several steps can be optimized to suit the individual needs of researchers. This procedure is robust and has consistently resulted in isolated mitochondria with above-average RCR values across animal models and tissue sources.

Author Spotlight: Uncovering the Role of Mitochondrial Calcium Phosphate in Heart Failure and Bioenergetics

Bioenergetic and metabolomic studies on mitochondria have revealed their multifaceted role in many diseases, but the isolation methods for these organelles vary. The method detailed here is capable of purifying high-quality mitochondria from multiple tissue sources. Quality is determined by respiratory control ratios and other metrics assessed with high-resolution respirometry.

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Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

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Biology

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Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

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Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

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Improved Protein Quantification Using Two-Step Mitochondrial Isolation

Two-Step Tag-Free Isolation of Mitochondria for Improved Protein Discovery and Quantification

Most physiological and disease processes, from central metabolism to immune response to neurodegeneration, involve mitochondria. The mitochondrial proteome is composed of more than 1,000 proteins, and the abundance of each can vary dynamically in response to external stimuli or during disease progression. Here, we describe a protocol for isolating high-quality mitochondria from primary cells and tissues. The two-step procedure comprises (1) mechanical homogenization and differential centrifugation to isolate crude mitochondria, and (2) tag-free immune capture of mitochondria to isolate pure organelles and eliminate contaminants. Mitochondrial proteins from each purification stage are analyzed by quantitative mass spectrometry, and enrichment yields are calculated, allowing the discovery of novel mitochondrial proteins by subtractive proteomics. Our protocol provides a sensitive and comprehensive approach to studying mitochondrial content in cell lines, primary cells, and tissues.

Author Spotlight: Two-Step Tag-Free Isolation of Mitochondria for Improved Protein Discovery and Quantification  

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

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Chapters in this video

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

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Rapid Isolation of the Mitoribosome from HEK Cells

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

Robust Mitochondrial Isolation from Rodent Cardiac Tissue

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Two-Step Tag-Free Isolation of Mitochondria for Improved Protein Discovery and Quantification