Name: isolation and respiratory measurements of mitochondria from arabidopsis thaliana
Uploaded: 2018-01-05T13:00:00
Description: Watch this Scientific Journal Video about Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana at JoVE.com

This study was supported by an Australian Research Council Centre of Excellence in Plant Energy Biology CE140100008, an Australian Research Council Future Fellowship (FT130100112) to MWM, and a Feodor Lynen Research Fellowship (Alexander von Humboldt Foundation, Germany) to JS.

This protocol is used for the isolation of intact mitochondria from Arabidopsis thaliana organs grown on soil using continuous colloidal density gradients. All procedures following the collection of the material are carried out at 4 &#176;C.
1. Preparation of Grinding Medium, Wash Buffer, and Gradient Solutions
<ol>
	<li>Prepare 300 mL of grinding medium (minus ascorbate and cysteine) and 200 mL of 2x wash buffer per Table 1 one day prior to the isolation, keep them...

<ol>
	<li>Day, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highlights+in+plant+mitochondrial+research.+Methods+in+molecular+biology.">Highlights in plant mitochondrial research. Methods in molecular biology.</a> Plant mitochondria. 1305, v-xvi (2015).</li><li>Millar, A. H., Carrie, C., Pogson, B., Whelan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+function-location+nexus:+Using+multiple+lines+of+evidence+in+defining+the+subcellular+location+of+plant+proteins.">Exploring the function-location nexus: Using multiple lines of evidence in defining the subcellular location of plant proteins.</a> Plant Cell. 21 (6), 1625-1631 (2009).</li><li>Eubel, H., 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=Free-flow+electrophoresis+for+purification+of+plant+mitochondria+by+surface+charge.">Free-flow electrophoresis for purification of plant mitochondria by surface charge.</a> Plant J. 52 (3), 583-594 (2007).</li><li>Murcha, M. W., 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=Protein+import+into+plant+mitochondria:+Signals,+machinery,+processing,+and+regulation.">Protein import into plant mitochondria: Signals, machinery, processing, and regulation.</a> J. Exp. Bot. 65 (22), 6301-6335 (2014).</li><li>Carrie, C., 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=Approaches+to+defining+dual-targeted+proteins+in+Arabidopsis.">Approaches to defining dual-targeted proteins in Arabidopsis.</a> Plant J. 57 (6), 1128-1139 (2009).</li><li>Pinto, G., Radulovic, M., Godovac-Zimmermann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+perspectives+in+the+redox+code+-+Mass+spectrometric+proteomics+studies+of+moonlighting+proteins.">Spatial perspectives in the redox code - Mass spectrometric proteomics studies of moonlighting proteins.</a> Mass Spectrom. Rev. , (2016).</li><li>Day, D. A., Neuburger, M., Douce, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+characterisation+of+chlorophyll-free+mitochondria+from+pea+leaves.">Biochemical characterisation of chlorophyll-free mitochondria from pea leaves.</a> Aust. J. Plant Physiol. 12 (3), 219-228 (1985).</li><li>Boersema, P. J., Raijmakers, R., Lemeer, S., Mohammed, S., Heck, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+peptide+stable+isotope+dimethyl+labeling+for+quantitative+proteomics.">Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics.</a> Nat. Protoc. 4 (4), 484-494 (2009).</li><li>Cheah, M. H., 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=Online+oxygen+kinetic+isotope+effects+using+membrane+inlet+mass+spectrometry+can+differentiate+between+oxidases+for+mechanistic+studies+and+calculation+of+their+contributions+to+oxygen+consumption+in+whole+tissues.">Online oxygen kinetic isotope effects using membrane inlet mass spectrometry can differentiate between oxidases for mechanistic studies and calculation of their contributions to oxygen consumption in whole tissues.</a> Anal Chem. 86 (10), 5171-5178 (2014).</li><li>Chrobok, D., 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=Dissecting+the+metabolic+role+of+mitochondria+during+developmental+leaf+senescence.">Dissecting the metabolic role of mitochondria during developmental leaf senescence.</a> Plant Physiol. 172 (4), 2132-2153 (2016).</li><li>Lee, C. P., Eubel, H., O'Toole, N., Millar, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+proteomics+of+root+and+shoot+mitochondria+and+transcript+analysis+to+define+constitutive+and+variable+components+in+plant+mitochondria.">Combining proteomics of root and shoot mitochondria and transcript analysis to define constitutive and variable components in plant mitochondria.</a> Phytochemistry. 72 (10), 1092-1098 (2011).</li><li>Lee, C. P., Eubel, H., Solheim, C., Millar, A. 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+proteome+heterogeneity+between+tissues+from+the+vegetative+and+reproductive+stages+of+Arabidopsis+thaliana+development.">Mitochondrial proteome heterogeneity between tissues from the vegetative and reproductive stages of Arabidopsis thaliana development.</a> J. Proteome Res. 11 (6), 3326-3343 (2012).</li><li>Millar, A. H., Whelan, J., Soole, K. L., Day, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+and+regulation+of+mitochondrial+respiration+in+plants.">Organization and regulation of mitochondrial respiration in plants.</a> Annu. Rev. Plant Biol. 62, 79-104 (2011).</li><li>Peters, 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=Complex+I+-+complex+II+ratio+strongly+differs+in+various+organs+of+Arabidopsis+thaliana.">Complex I - complex II ratio strongly differs in various organs of Arabidopsis thaliana.</a> Plant Mol. Biol. 79 (3), 273-284 (2012).</li><li>Werhahn, W. H., 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=Purification+and+characterization+of+the+preprotein+translocase+of+the+outer+mitochondrial+membrane+from+Arabidopsis+thaliana:+Identification+of+multiple+forms+of+TOM20.">Purification and characterization of the preprotein translocase of the outer mitochondrial membrane from Arabidopsis thaliana: Identification of multiple forms of TOM20.</a> Plant Physiol. 125 (2), 943-954 (2001).</li><li>Heazlewood, J. L., Howell, K. A., Whelan, J., Millar, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+an+analysis+of+the+rice+mitochondrial+proteome.">Towards an analysis of the rice mitochondrial proteome.</a> Plant Physiol. 132 (1), 230-242 (2003).</li></ol>

Typically, isolation of mitochondria from Arabidopsis leaves yields up 3 mg of mitochondria from approximately 80 - 100 3 - 4-week old plants, although yields of greater than 5 mg can often be achieved with thorough grinding. The yield varies with growth conditions and decreases dramatically as leaves senesce, although mitochondria structure seems to be well maintained during senescence9. One of the most critical features to obtain a good yield is the method of grinding to lyse cells to release mi...

The history of plant mitochondrial research goes back over 100 years1. Intact mitochondria were first isolated in the early 1950s using differential centrifugation. The advent of a colloidal density gradient in the 1980s allowed mitochondria to be purified without suffering osmotic adjustment. While gradient purified mitochondria are suitable for most purposes, due to the sensitivity of mass spectrometry, even relatively minor contaminants can be detected and may be inappropriately assigned a mitochondrial location2. The use of free flow electrophoresis can remove both plastidic and peroxisome contamination3, but free flow electrophoresis is a highly specialized technique and is not required for the vast majority of studies. Furthermore, when determining the location of a protein it needs to be remembered that dual or multiple targeting of proteins occurs in cells. Over 100 dual targeted proteins are described for chloroplasts/plastids and mitochondria4, and a number of proteins targeted to mitochondria and peroxisomes are also known5. Furthermore, the re-location of proteins under specific stimuli, e.g. oxidative stress, is an emerging theme in cell biology6. Thus, the location of proteins needs to be considered in the context of the biology studied, and a variety of approaches are used to determine and verify location2.
Mitochondria are typically isolated from plant tissues by homogenization, a balance is required between breaking open the cell wall to release mitochondria, and not damaging the mitochondria. Traditionally, with potato and cauliflower, homogenization involves using household blender/juicer apparatus to make a liquid extract in a buffer with various components to maintain activity.&#160;Isolation of mitochondria from pea leaves, (a popular material for mitochondrial isolation using young seedlings (~10 days old), utilizes a blender to lyse cells as the leaf material is soft. With the availability of Arabidopsis thaliana T-DNA insertional knock-out lines, the need to be able to purify mitochondria to carry out functional studies has necessitated the development of methods to isolate mitochondria from leaf, root or flower tissue. Overall the methods developed for other plants worked well7, with the perquisite that grinding of the material needed to be optimized. For Arabidopsis this can be achieved in a variety of ways (see below), and differs between tissue types (root versus shoot). The use of the continuous gradient can also be optimized as the density of mitochondria from different organs or developmental stages means they can migrate differently. Thus, for maximum separation the density of the gradient can be refined to ensure to achieve best separation.
Once purified the mitochondria can be used for a variety of studies, including protein and tRNA uptake experiments, enzyme activity assays, respiratory chain measurements and western blot analyses. Isolated mitochondria can also be used for mass spectrometry analyses of protein abundance. Targeted multiple reaction monitoring (MRM) analyses allows for the quantification of defined proteins, but require significant assay development. In contrast, quantification by dimethyl or other isotope labels8, provides a discovery approach in identifying differences across the whole proteome that can be used to uncover novel biological insights.

<table>
	<tbody>
		<tr>
			<td>ADP</td>
			<td>Sigma-Aldrich</td>
			<td>A2754</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>Sigma-Aldrich</td>
			<td>A8674</td>
			<td>Chemical, dissolve in ethanol</td>
		</tr>
		<tr>
			<td>AOX antibody</td>
			<td>from Tom Elthon</td>
			<td></td>
			<td>Elthon et al., 1989</td>
		</tr>
		<tr>
			<td>Ascorbate</td>
			<td>Sigma-Aldrich</td>
			<td>A0157</td>
			<td>Ascorbate Oxidase from Cucurbita sp.</td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma-Aldrich</td>
			<td>A26209</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Bovogen</td>
			<td>BSAS 1.0</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Clarity western ECL substrate</td>
			<td>Bio-Rad Laboratories</td>
			<td>1705061</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Criterion Stain-Free Precast Gels 8-16% 18 Wells</td>
			<td>Bio-Rad Laboratories</td>
			<td>5678104</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Cyanide</td>
			<td>Sigma-Aldrich</td>
			<td>60178</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Cytochrome c</td>
			<td>Sigma-Aldrich</td>
			<td>C3131</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Difco Agar, granulated</td>
			<td>BD Biosciences</td>
			<td>214530</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Dithiotreitol</td>
			<td>Sigma-Aldrich</td>
			<td>D0632</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>EDTA disodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>E5134</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Gamborg B-5 Basal Medium</td>
			<td>Austratec</td>
			<td>G398-100L</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Gamborg Vitamin Solution (1000x)</td>
			<td>Austratec</td>
			<td>G219-100ML</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG (H + L)-HRP Conjugate</td>
			<td>Bio-Rad Laboratories</td>
			<td>1706516-2ml</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit IgG (H + L)-HRP Conjugate</td>
			<td>Bio-Rad Laboratories</td>
			<td>1706515-2ml</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma</td>
			<td>C7352-100G</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>230391</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Murashige &#38; Skoog Basal Salt Mixture (MS)</td>
			<td>Austratec</td>
			<td>M524-100L</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Myxothiazol</td>
			<td>Sigma-Aldrich</td>
			<td>T5580</td>
			<td>Chemical, dissolve in ethanol</td>
		</tr>
		<tr>
			<td>NADH</td>
			<td>Sigma-Aldrich</td>
			<td>N8129</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Ndufs4 antibody</td>
			<td>from Etienne Meyer</td>
			<td></td>
			<td>Meyer et al., 2009</td>
		</tr>
		<tr>
			<td>n-Propyl gallate</td>
			<td>Sigma-Aldrich</td>
			<td>P3130</td>
			<td>Chemical, dissolve in ethanol</td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>GE Healthcare</td>
			<td>17-0891-01</td>
			<td>Chemical, colloidal density gradient</td>
		</tr>
		<tr>
			<td>Polyvinylpyrrolidone (PVP40)</td>
			<td>Sigma-Aldrich</td>
			<td>PVP40</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Potassium cyanide</td>
			<td>Sigma-Aldrich</td>
			<td>60178</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (KH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P2256</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Chem-Supply</td>
			<td>SA046</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Sodium dithionite</td>
			<td>Sigma-Aldrich</td>
			<td>157953</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Sodium L-ascorbate</td>
			<td>Sigma</td>
			<td>A4034-100G</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Succinate</td>
			<td>Sigma-Aldrich</td>
			<td>S2378</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Chem-Supply</td>
			<td>SA030</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>TES</td>
			<td>Sigma-Aldrich</td>
			<td>T1375</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Tetrasodium pyrophosphate (Na4P2O7 &#183; 10H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>221368</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Trans-Blot Turbo RTA Midi Nitrocellulose Transfer Kit</td>
			<td>Bio-Rad Laboratories</td>
			<td>1704271</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Triton-X 100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td>Chemical, detergent</td>
		</tr>
		<tr>
			<td>Western Blocking Reagent</td>
			<td>Sigma</td>
			<td>11921681001</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Balance</td>
			<td>Mettler Toledo</td>
			<td>XS204</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Beakers</td>
			<td>Isolab</td>
			<td>50 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>Avanti J-26XP</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Centrifuge tubes</td>
			<td>Nalgene</td>
			<td>3117-9500</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Circulator</td>
			<td>Julabo</td>
			<td>1124971</td>
			<td>Attached to oxygen electrode chamber</td>
		</tr>
		<tr>
			<td>Conical flask</td>
			<td>Isolab</td>
			<td>500 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Dropper</td>
			<td></td>
			<td>3 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixed angle rotor</td>
			<td>Beckman Coulter</td>
			<td>JA25.5</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Funnel</td>
			<td>Per Alimenti</td>
			<td>14 cm</td>
			<td>For filtering</td>
		</tr>
		<tr>
			<td>Gradient pourer</td>
			<td>Bio-Rad</td>
			<td>165-4120</td>
			<td>For preparation of gradients</td>
		</tr>
		<tr>
			<td>Magnetic Stirrer ATE</td>
			<td>VELP Scientifica</td>
			<td>F20300165</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Miracloth</td>
			<td>VWR</td>
			<td>EM475855-1R</td>
			<td>Filtration material</td>
		</tr>
		<tr>
			<td>Mortar and pestle</td>
			<td>Jamie Oliver</td>
			<td>Granite, 6 Inch</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>O2view</td>
			<td>Hansatech Instruments</td>
			<td></td>
			<td>Oxygen monitoring software</td>
		</tr>
		<tr>
			<td>Oxygraph Plus System</td>
			<td>Hansatech Instruments</td>
			<td>1187253</td>
			<td>Clark-type oxygen electrode</td>
		</tr>
		<tr>
			<td>Paintbrush</td>
			<td>Artist first choice</td>
			<td>1008R-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM-996</td>
			<td>plastic paraffin film</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Gilson</td>
			<td>F155001</td>
			<td>For preparation of gradients</td>
		</tr>
		<tr>
			<td>PVC peristaltic tubing</td>
			<td>Gilson</td>
			<td>F117930</td>
			<td>For preparation of gradients</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>VELP Scientifica</td>
			<td>OCB</td>
			<td>Equipment</td>
		</tr>
	</tbody>
</table>

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.

Using this protocol, we were able to detect different mitochondrial proteins by SDS-PAGE and immunoblotting. As shown in Figure 3A, the protein isolated from water culture tissue is sufficient to detect a faint band (2 &#181;g). Signal intensity increases proportionately to the amounts loaded. For mitochondria isolated from tissues grown on plates (Figure 3B), the response to high light stress treatment in different genotypes can...

Watch this Scientific Journal Video about Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana at JoVE.com

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.

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 ...

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Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as ...

Isolation of Tissue Extracellular Vesicles from the Liver

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in ...

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 ...

Mitochondria and Endoplasmic Reticulum Imaging by Correlative Light and Volume Electron Microscopy

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved ...

Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and ...