Diese Arbeit wurde durch Zuschüsse aus dem CIHR und der NSERC of Canada unterstützt. VIT ist ein CIHR New Investigator und Concordia University Research Chair in Genomics, Cell Biology and Aging.

Material und Methoden Hefestämme und Wachstumsbedingungen Der Wildtypstamm BY4742 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) wurde in den reichen YEPD Medium (1% Hefeextrakt, 2% Pepton, 2% Glucose). Die Zellen wurden bei 30 ° C mit Rotations-Schütteln bei 200 rpm in Erlenmeyer-Kolben bei einer &quot;Flasche Volumen / Medium Volume&quot;-Verhältnis von 5:1 kultiviert. Isolation von Roh-mitochondriale Fraktion <ol><li> Grow 1 L der Wildtypstamm BY4742 Kultur für 48 h </li><li> Gießen Kultur in 500-ml Nalgene Zentrifugenröhrchen. Balance der Last und Ernte Zellen durch Zentrifugation für 5 min bei 3000 xg bei Raumtemperatur. </li><li> Dekantieren Überstand und resuspendieren pelletierten Zellen in 250 ml dH2O. Pellet-Zellen durch Zentrifugation für 5 min bei 3000 xg bei Raumtemperatur. </li><li> Dekantieren Überstand und resuspendieren pelletierten Zellen in 250 ml dH2O. Pellet-Zellen durch Zentrifugation für 5 min bei 3000 xg bei Raumtemperatur. </li><li> Bestimmen Sie Nassgewicht Zellpellet. </li><li> Resuspendieren pelletierten Zellen in DTT-Puffer [2 ml Puffer / g (Feuchtgewicht) Zellen]. </li><li> Übertragen Sie die Zellsuspension auf 50-ml Falcon Rohre aus Kunststoff. </li><li> Drehen Sie das Röhrchen bei 70 rpm in einem Schüttler für 20 min bei 30 ° C. </li><li> Ernten Sie die Zellen durch Zentrifugation für 5 min bei 3000 xg bei Raumtemperatur. </li><li> Resuspendieren pelletierten Zellen in Zymolyase Puffer ohne Zymolyase [7 ml Puffer / g (Feuchtgewicht) Zellen]. </li><li> Pellet-Zellen durch Zentrifugation für 5 min bei 3000 xg bei Raumtemperatur. </li><li> Resuspendieren pelletierten Zellen in Zymolyase Puffer ohne Zymolyase [7 ml Puffer / g (Feuchtgewicht) Zellen]. </li><li> Transfer-Zell-Suspension in einem Glaskolben. </li><li> Fügen Sie die Pulver von Zymolyase-100T [1 mg Zymolyase-100T / g (Feuchtgewicht) Zellen], um die Zell-Suspension. </li><li> Drehen Sie den Kolben mit Zellsuspension bei 70 rpm in einem Schüttler für 30 min bei 30 ° C. </li><li> Transfer Sphäroplasten gebildet durch die Verdauung der Zellwand mit Zymolyase-100T bis 50-ml Kunststoff-Röhrchen. </li><li> Pellet Sphäroplasten durch Zentrifugation für 8 min bei 2200 xg bei 4 ° C. </li></ol> ** Alle weiteren Schritte sollten bei 4 ° C oder auf Eis durchgeführt werden. Suspensionen von Sphäroplasten sollten schonend behandelt, mit Pipetten mit geschnittenen Spitzen zu vermeiden brechen Organellen werden .** <ol start="18"><li> Resuspendieren pelletiert Sphäroplasten in eiskaltem Homogenisierungspuffer [6,5 ml Puffer / g (Feuchtgewicht) Zellen]. </li><li> Pellet Sphäroplasten durch Zentrifugation für 8 min bei 2200 xg bei 4 ° C. </li><li> Resuspendieren pelletiert Sphäroplasten in eiskaltem Homogenisierungspuffer [6,5 ml Puffer / g (Feuchtgewicht) Zellen]. </li><li> Transfer-Zellen zu einer pre-auf Eis gekühlt Glashomogenisator. </li><li> Mit einer engen Pistill Homogenisierung der Zellen, indem sie 15 Schläge von dem Pistill. </li><li> Add 1 Volumen eiskaltem Homogenisierungspuffer. </li><li> Transfer homogenisiert Sphäroplasten bis 50-ml Kunststoff-Röhrchen. </li><li> Pellet ungebrochene Zellen, Kerne und große Trümmer durch Zentrifugation für 5 min bei 1500 xg bei 4 ° C. </li><li> Centrifuge der resultierende Überstand für 5 min bei 3000 xg bei 4 ° C. </li><li> Centrifuge der resultierende Überstand für 15 min bei 12000 xg bei 4 ° C. </li><li> Dekantieren und Pellet in eiskaltem Homogenisierungspuffer [6,5 ml Puffer / g (Feuchtgewicht) Zellen]. </li><li> Zentrifugieren Sie für 5 min bei 3000 xg bei 4 ° C. </li><li> Centrifuge der resultierende Überstand für 15 min bei 12000 xg bei 4 ° C. </li><li> Dekantieren und Pellet in 3 ml eiskaltem SEM-Puffer. </li></ol> ** Obwohl diese Suspension in den Mitochondrien angereichert ist, es enthält auch andere Organellen wie das endoplasmatische Retikulum (Mikrosomen), Golgi und Vakuolen. Um reine Mitochondrien, können diese rohe mitochondriale Fraktion zur weiteren Fraktionierung unterworfen werden, wie unten beschrieben .** Reinigung von Mitochondrien ohne Verunreinigung durch andere Organellen <ol><li> Legen Sie 1,5 ml eiskaltem 60% (w / v) Saccharose in EM-Puffer in ein Beckman Ultra-Clear-Zentrifugenröhrchen. </li><li> Overlay 60% (w / v) Saccharose mit 4 ml 32%, 1,5 ml von 23%, 1,5 ml 15% Saccharose (alle wt / v in EM-Puffer). </li><li> Platz 3 ml des Roh-mitochondriale Fraktion in SEM-Puffer auf 15% (w / v) Saccharose. </li><li> Zentrifuge in einem Beckman SW41 Ti Ausschwingrotor Rotor für 1 h bei 134000 xg (33000 rpm) bei 4 ° C. </li><li> Die intakten Mitochondrien bilden ein braunes Band an der 60% / 32% Saccharose-Schnittstelle. </li><li> Entfernen Sie vorsichtig Saccharose bis zum Erreichen der mitochondrialen Band. </li><li> Entfernen Sie die mitochondriale Band mit einer Pipette mit einem Cut-Spitze und legen Sie sie in ein Beckman Zentrifugenröhrchen für ein MLS-5 Rotor. </li><li> Füllen Sie das Rohr mit SEM-Puffer. </li><li> Pellet der reinen Mitochondrien durch Zentrifugation in einem MLS-5-Rotor für 30 min bei 10000 xg (31000 rpm) für 30 Minuten bei 4 ° C. </li><li> Dekantieren Überstand miteiner Pipette. </li><li> Verwenden Sie die reine Mitochondrien für die Analyse der mitochondrialen Funktionen, mitochondriale Proteom-und lipidome, und die mitochondriale DNA. </li></ol> Reagenzien <ol><li> DTT-Puffer [100 mM Tris/H2SO4 (pH 9,4), 10 mM Dithiothreitol] (für 15 ml) <ul style="list-style-type:none;"><li> 1,5 ml 1 M Tris/H2SO4 Puffer (pH 9,4) </li><li> 150 ul 1 M DTT </li><li> Volume auf 15 ml </li></ul></li><li> Zymolyase Puffer [20 mM Kaliumphosphat (pH 7,4), 1,2 M Sorbitol] (für 100 ml) <ul style="list-style-type:none;"><li> 2 ml 1 M Kaliumphosphatpuffer (pH 7,4) </li><li> 60 ml 2 M Sorbit </li><li> Volumen auf 100 ml </li></ul></li><li> Homogenisierungspuffer [10 mM Tris / HCl (pH 7,4), 0,6 M Sorbitol, 1 mM EDTA, 0,2% (w / v) BSA] (für 250 ml] <ul style="list-style-type:none;"><li> 2,5 ml 1 M Tris / HCl-Puffer (pH 7,4) </li><li> 75 ml 2 M Sorbit </li><li> 500 ul 500 mM EDTA </li><li> 0,5 g BSA </li><li> Volume auf 250 ml </li></ul></li><li> SEM-Puffer [10 mM MOPS / KOH (pH 7,2), 250 mM Saccharose, 1 mM EDTA] (für 250 ml) <ul style="list-style-type:none;"><li> 2,5 ml 1 M MOPS / KOH-Puffer (pH 7,2) </li><li> 31,25 ml 2 M Saccharose </li><li> 500 ul 500 mM EDTA </li><li> Volume auf 250 ml </li></ul></li><li> EM-Puffer [10 mM MOPS / KOH (pH 7,2), 1 mM EDTA] (für 250 ml) <ul style="list-style-type:none;"><li> 2,5 ml 1 M MOPS / KOH-Puffer (pH 7,2) </li><li> 500 ul 500 mM EDTA </li><li> Volume auf 250 ml </li></ul></li></ol>

<ol>
	<li>Voet, D., Voet, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemistry.">Biochemistry.</a> , (2004).</li><li>Suen, D. -. F., Norris, K. L., Youle, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dynamics+and+apoptosis.">Mitochondrial dynamics and apoptosis.</a> Genes Dev. 22, 1577-1590 (2008).</li><li>Cheng, W. C., Berman, S. B., Ivanovska, I., Jonas, E. A., Lee, S. J., Chen, Y., Kaczmarek, L. K., Pineda, F. &. a. m. p. ;. a. m. p., Hardwick, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+factors+with+dual+roles+in+death+and+survival.">Mitochondrial factors with dual roles in death and survival.</a> Oncogene. 25, 4697-4705 (2006).</li><li>Detmer, S. A., Chan, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+and+dysfunctions+of+mitochondrial+dynamics.">Functions and dysfunctions of mitochondrial dynamics.</a> Nat. Rev. Mol. Cell Biol. 8, 870-879 (2007).</li><li>McBride, H. M., Neuspiel, M., Wasiak, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria:+more+than+just+a+powerhouse.">Mitochondria: more than just a powerhouse.</a> Curr. Biol. 16, R551-R560 (2006).</li><li>Balaban, R. S., Nemoto, S., Finkel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria,+oxidants,+and+aging.">Mitochondria, oxidants, and aging.</a> Cell. 120, 483-495 (2005).</li></ol>

The authors have nothing to disclose.

Dieses Verfahren ermöglicht die High-Yield-Isolierung reiner Mitochondrien aus Hefezellen. Mitochondrien mit dieser Methode gereinigt sind im Wesentlichen frei von Verunreinigungen durch andere Organellen und behalten ihre strukturelle und funktionelle Integrität nach ihrer Reinigung. Die beschriebene Methode liefert Mitochondrien, die sich für zellfreie Rekonstitution wesentlicher mitochondriale Prozesse sind. Diese hochreinen Mitochondrien können auch für die Analyse der mitochondrialen Struktur und Funktionen, m...

purification of mitochondria from yeast cells

Mitochondrien sind die Haupt-Website des ATP-Produktion im aeroben Stoffwechsel in eukaryotischen Zellen nicht-photosynthetischen<sup&gt; 1</sup&gt;. Diese komplexen Organellen spielen auch eine wesentliche Rolle im apoptotischen Zelltod<sup&gt; 2</sup&gt;, Das Überleben der Zelle<sup&gt; 3</sup&gt; Entwicklung von Säugetieren<sup&gt; 4</sup&gt;, Neuronale Entwicklung und Funktion<sup&gt; 4</sup&gt;, Intrazelluläre Signalwege<sup&gt; 5</sup&gt; Und Langlebigkeit Regulierung<sup&gt; 6</sup&gt;. Unser Verständnis dieser komplexen biologischen Prozesse durch Mitochondrien gesteuert setzt auf robuste Methoden zur Beurteilung ihrer Morphologie, ihrem Protein-und Lipid-Zusammensetzung, die Integrität ihrer DNA, und ihre zahlreichen lebenswichtigen Funktionen. Die Bierhefe<em&gt; Saccharomyces cerevisiae</em&gt;, Ist eine genetisch und biochemisch manipulierbar einzelligen Eukaryoten mit annotierten Genom-und Proteom-wohldefinierte, ein wertvolles Modell für die Erforschung der molekularen und zellulären Mechanismen, die die wesentlichen biologischen Funktionen der Mitochondrien. Für diese Arten von Studien, ist es entscheidend, hochreinen Mitochondrien haben. Hier präsentieren wir Ihnen eine detaillierte Beschreibung der eine schnelle und effektive Methode zur Reinigung von Hefe-Mitochondrien. Diese Methode ermöglicht die Isolierung von hochreinem Mitochondrien, die im Wesentlichen frei von Verunreinigungen durch andere Organellen sind und behalten ihre strukturelle und funktionelle Integrität nach ihrer Reinigung. Mitochondrien mit dieser Methode gereinigt werden für die zellfreie Rekonstitution wesentlicher mitochondriale Prozesse geeignet und können für die Analyse der mitochondrialen Struktur und Funktionen, mitochondriale Proteom-und lipidome und mitochondriale DNA verwendet werden.

Watch this Scientific Journal Video about Purification of Mitochondria from Yeast Cells at JoVE.com

Purification of Mitochondria from Yeast Cells

Wir beschreiben eine schnelle und effektive Methode zur Aufreinigung von Mitochondrien aus der Hefe<em&gt; Saccharomyces cerevisiae</em&gt;. Dieses Verfahren ermöglicht die High-Yield-Isolierung reiner Mitochondrien, die im Wesentlichen frei von Verunreinigungen durch andere Organellen sind und behalten ihre strukturelle und funktionelle Integrität nach ihrer Reinigung.

Reinigung von Mitochondrien aus Hefezellen

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

purification-of-mitochondria-from-yeast-cells

Research

JoVE Journal

Biology

24.9K Aufrufe.  Concordia University. Wir beschreiben eine schnelle und effektive Methode zur Aufreinigung von Mitochondrien aus der Hefe Saccharomyces cerevisiae. Dieses Verfahren ermöglicht die High-Yield-Isolierung reiner Mitochondrien, die im Wesentlichen frei von Verunreinigungen durch andere Organellen sind und behalten ihre strukturelle und funktionelle Integrität nach ihrer Reinigung.

Serie: Purification of Mitochondria from Yeast Cells

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

Isolierung von CD4 + T-Zellen aus Maus-Lymphknoten mit Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

Forschung

Biologie

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Färbung von Proteinen in Gelen mit Coomassie G-250 ohne organische Lösungsmittel und Essigsäure

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Reinigung von Vorläufern aus Skelettmuskeln

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

Chromatographische Reinigung von hoch aktiven Hefe Ribosomen

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

Die konfokale Bildgebung von Einzel Mitochondrial Super Blinkt Intact Herz oder<em&gt; In Vivo</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized near the mitochondria. Support for this possibility is derived from recent studies, in which many mRNAs encoding mitochondrial proteins were shown to be localized to the mitochondria vicinity. Together with earlier demonstrations of ribosomes&#8217; association with the outer membrane, these results suggest a localized translation process. Such localized translation may improve import efficiency, provide unique regulation sites and minimize cases of ectopic expression. Diverse methods have been used to characterize the factors and elements that mediate localized translation. Standard among these is subcellular fractionation by differential centrifugation. This protocol has the advantage of isolation of mRNAs, ribosomes and proteins in a single procedure. These can then be characterized by various molecular and biochemical methods. Furthermore, transcriptomics and proteomics methods can be applied to the resulting material, thereby allow genome-wide insights. The utilization of yeast as a model organism for such studies has the advantages of speed, costs and simplicity. Furthermore, the advanced genetic tools and available deletion strains facilitate verification of candidate factors.

Many mRNAs encoding mitochondrial proteins are associated with the mitochondria outer membrane. We describe a subcellular fractionation procedure aimed at isolation of yeast mitochondria with its associated mRNAs and ribosomes. This protocol can be applied to cells grown under diverse conditions&#160;in order to reveal mechanisms of mRNA localization and localized translation near the mitochondria.

Isolierung von mRNAs Verbunden mit Hefe-Mitochondrien Study Mechanismen der lokalisierten Übersetzung

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized ...

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.

Schnelle Isolierung und Reinigung von Mitochondrien für die Transplantation von Gewebe-Dissoziation und Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

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

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.

Isolierung und funktionale Analyse von Mitochondrien aus kultivierten Zellen und Maus-Gewebe

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

Genomweite Reinigung von extrachromosomale zirkuläre DNA aus eukaryotischen Zellen

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...

Purification of Platelets from Mouse Blood

Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), and plasma proteins. We describe here purification of platelets from mouse blood using three-fold more iohexol gradient medium relative to blood sample volume and centrifugation in a swinging bucket rotor at 400 x g for 20 min at 20 &#176;C. The recovery/yield of the purified platelets were 18.2-38.5%, and the purified platelets were in a resting state, which did not contain any significant number of RBC and WBC. The purified platelets treated with thrombin showed up to 93% activation, indicating their viability. We confirmed that the purified platelets are sufficiently pure using flow cytometric and microscopic evaluation. These platelets can be used for gene expression, activation, granule release, aggregation, and adhesion assays. This method can be used for purification of platelets from the blood of other species as well.

Here, mouse blood was collected in the presence of an anti-coagulant. The platelets were purified by iohexol gradient medium using low speed centrifugation. The platelets were activated with thrombin to investigate if they were viable. The quality of the purified platelets was analyzed by flow cytometry and microscopy.

Reinigung von Plateletten aus dem Mausblut

Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), ...