이 작품은 CIHR과 캐나다의 NSERC에서 보조금에 의해 지원되었다. VIT는 CIHR 새로운 인베스티게이터와 게놈의 콩코디아 대학 연​​구 위원장, 세포 생물학 및 노화입니다.

재료 및 방법 효모 변종과 성장 조건 야생 형 변형 BY4742는 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) (1 % 효모 추출물, 펩톤 2 %, 2 % 포도당) 풍부한 YEPD 매체에서 재배되었다. 전지는 30 ° C 회전이 5시 1분의 &quot;플라스크 볼륨 / 볼륨 매체&quot;비율 Erlenmeyer flasks의 200 rpm으로 잡고 교양되었습니다. 원유 mitochondrial 분획의 분리 <ol><li> 48 H.위한 야생 형 스트레인 BY4742 문화 1 L 그로 </li><li> 500 ML Nalgene의 원심 분리기 튜브의 문화를 하거라. 상온에서 3,000 XG에서 5 분 원심 분리하여 부하와 수확 세포를 균형. </li><li> dH2O 250 ML에 가만히 따르다 뜨는 및 resuspend pelleted 세포. 상온에서 3,000 XG에서 5 분 원심 분리하여 세포 펠렛. </li><li> dH2O 250 ML에 가만히 따르다 뜨는 및 resuspend pelleted 세포. 상온에서 3,000 XG에서 5 분 원심 분리하여 세포 펠렛. </li><li> 세포 펠렛의 서부 유럽 표준시 무게를 결정합니다. </li><li> DTT 버퍼 [버퍼 / G (서부 유럽 표준시 무게) 전지 2 ML]에서 pelleted 세포를 Resuspend. </li><li> 50 ML 팰컨 플라스틱 튜브에 세포 현탁액을 전송합니다. </li><li> 30 ° C. 20 분 쉐이크 70 RPM에서 튜브를 회전 </li><li> 상온에서 3,000 XG에서 5 분 원심 분리하여 세포를 수확. </li><li> Zymolyase [버퍼 / G (서부 유럽 표준시 무게) 전지 7 ML]없이 Zymolyase 버퍼에 pelleted 세포를 Resuspend. </li><li> 상온에서 3,000 XG에서 5 분 원심 분리하여 세포 펠렛. </li><li> Zymolyase [버퍼 / G (서부 유럽 표준시 무게) 전지 7 ML]없이 Zymolyase 버퍼에 pelleted 세포를 Resuspend. </li><li> 유리 플라스크에 세포 현탁액을 전송합니다. </li><li> Zymolyase - 100T의 분말을 추가 [1 Zymolyase - 100T / G (서부 유럽 표준시 무게) 세포의 MG] 세포 현탁액 수 있습니다. </li><li> 30 ° C. 30 분 쉐이크 70 rpm으로 세포 현탁액으로 술병 회전 </li><li> 전송 spheroplasts 인해 Zymolyase - 100T 50 - ML의 플라스틱 원심 분리기 튜브와 세포 벽의 소화에 형성했다. </li><li> 펠렛 4 2,200 XG 8 분 원심 분리하여 spheroplasts ° C. </li></ol> ** 모든 후속 단계 ° C 또는 얼음 4 시에 실시한다. spheroplasts의 Suspensions는 부드럽게 organelles을 위반하지 않도록 컷 팁을 pipettes를 사용하여 처리되어야합니다 .** <ol start="18"><li> 얼음처럼 차가운 균질화 버퍼 [버퍼 / G (서부 유럽 표준시 무게) 세포의 6.5 ML]에서 pelleted spheroplasts을 Resuspend. </li><li> 펠렛 4 2,200 XG 8 분 원심 분리하여 spheroplasts ° C. </li><li> 얼음처럼 차가운 균질화 버퍼 [버퍼 / G (서부 유럽 표준시 무게) 세포의 6.5 ML]에서 pelleted spheroplasts을 Resuspend. </li><li> 얼음 유리 균질 화기에 미리 차게하기 위해 세포를 전송합니다. </li><li> 꽉 유봉을 사용하여 유봉 15 스트로크를 만들어 세포를 균질. </li><li> 얼음처럼 차가운 균질화 버퍼 1 볼륨을 추가합니다. </li><li> 50 ML의 플라스틱 원심 튜브에 균질 spheroplasts를 전송합니다. </li><li> 펠렛 4 1,500 XG에서 5 분 centrifuging에 의해 손상되지 않은 세포, 핵, 대형 부스러기 ° C. </li><li> 4 3,000 XG에서 5 분 결과 뜨는을 원심 분리기 ° C. </li><li> 4 ° C.를 XG 12,000 15 분 결과 뜨는을 원심 분리기 </li><li> 얼음처럼 차가운 균질화 버퍼에 가만히 따르다 뜨는 및 resuspend 펠렛 [버퍼 / G (서부 유럽 표준시 무게) 세포의 6.5 ML]. </li><li> 4 3,000 XG에서 5 분 원심 분리기 ° C. </li><li> 4 ° C.를 XG 12,000 15 분 결과 뜨는을 원심 분리기 </li><li> 얼음처럼 차가운 SEM 버퍼 3 ML에 가만히 따르다 뜨는 및 resuspend 펠릿. </li></ol> **이 정지가 mitochondria에 풍부한이지만,이 또한 endoplasmic reticulum (microsomes), 잠돌군 Zz 및 vacuoles 같은 다른 organelles가 포함되어 있습니다. 순수 mitochondria를 얻으려면,이 원유 mitochondrial 분획은 아래 설명한 바와 같이, 추가 분류를 받게 될 수 있습니다 .** 다른 organelles에 의해 오염의 mitochondria 찾아볼 수의 정화 <ol><li> 베크맨 울트라 클리어 원심 튜브에 EM 버퍼에 얼음처럼 차가운 60 % 플레이스 1.5 ML (W / V)은 자당. </li><li> 오버레이 60% (W / V) 32 %의 4 ML과 자당, 23 %의 1.5 ML 15 %의 자당 (모든 EM에서 WT / V 버퍼)의 1.5 ML. </li><li> 15 % (W / V) 자당 위에 SEM 버퍼에 원유 mitochondrial 분수 3 ML 놓습니다. </li><li> 4 134,000 XG (33,000 RPM) 1 H ° C.에 대한 베크맨 SW41 티 스윙 - 양동이 로터의 원심 분리기 </li><li> 60 % / 32 % 자당 인터페이스에서 그대로 mitochondria 형태는 갈색 밴드. </li><li> 조심스럽게 mitochondrial 밴드에 도달하기 전까지 자당를 제거합니다. </li><li> 절단 팁과 피펫을 사용하여 mitochondrial 밴드를 제거하고 MLS - 5 로터에 대한 베크맨 원심 관에 넣습니다. </li><li> SEM 버퍼로 튜브를 입력합니다. </li><li> 펠렛 4 30 분 10,000 XG (31,000 RPM)에서 30 분 MLS - 5 로터에서 원심 분리하여 순수 mitochondria ° C. </li><li> 사용 가만히 따르다 뜨는피펫. </li><li> mitochondrial 기능, mitochondrial 프로테옴 및 lipidome하고, mitochondrial DNA의 분석을위한 순수 mitochondria를 사용합니다. </li></ol> 시약 <ol><li> DTT 버퍼 [100 MM Tris/H2SO4 (산도 9.4), 10 MM dithiothreitol] (15 ML 용) <ul style="list-style-type:none;"><li> 1 M Tris/H2SO4 버퍼의 1.5 ML (산도 9.4) </li><li> 1 M DTT 150 μl </li><li> 15 ML에 볼륨 </li></ul></li><li> Zymolyase 버퍼 [20 MM의 칼륨 나트륨 (산도 7.4), sorbitol 1.2 M] (100 ML에 대한) <ul style="list-style-type:none;"><li> 1 M의 칼륨 인산 버퍼 2 ML (산도 7.4) </li><li> sorbitol이 M 60 ML </li><li> 100 ML에 볼륨 </li></ul></li><li> 균질화 버퍼 [10 MM 트리스 / HCL (산도 7.4), sorbitol 0.6 M, 1 ㎜ EDTA (에틸렌 다이아 민 테트라 초산), 0.2 % (W / V) BSA] (250 ML에 대한] <ul style="list-style-type:none;"><li> 1 M 트리스 / HCL 버퍼 (산도 7.4) 2.5 ML </li><li> sorbitol이 M의 75 ML </li><li> 500 MM EDTA (에틸렌 다이아 민 테트라 초산) 500 μl </li><li> BSA 0.5 g </li><li> 250 ML에 볼륨 </li></ul></li><li> SEM 버퍼 [10 MM의 걸레 / 코 (산도 7.2), 250 MM의 자당, 1 MM EDTA (에틸렌 다이아 민 테트라 초산)] (250 ML에 대한) <ul style="list-style-type:none;"><li> 1 M의 걸레 / 코 버퍼 (산도 7.2) 2.5 ML </li><li> 2 M의 자당의 31.25 ML </li><li> 500 MM EDTA (에틸렌 다이아 민 테트라 초산) 500 μl </li><li> 250 ML에 볼륨 </li></ul></li><li> EM 버퍼 [10 MM의 걸레 / 코 (산도 7.2), 1 MM EDTA (에틸렌 다이아 민 테트라 초산)] (250 ML에 대한) <ul style="list-style-type:none;"><li> 1 M의 걸레 / 코 버퍼 (산도 7.2) 2.5 ML </li><li> 500 MM EDTA (에틸렌 다이아 민 테트라 초산) 500 μl </li><li> 250 ML에 볼륨 </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.

이 방법은 효모 세포에서 순수 mitochondria의 높은 수율 격리 수 있습니다. 이 방법에 의해 정화 Mitochondria 다른 organelles에 의해 오염의 기본적 자유 및 정화 후 자신의 구조와 기능 무결성을 유지합니다. 필수 mitochondrial 프로세스 셀 - 무료 reconstitution에 적합 mitochondria 설명한 방법 산출. 이 높은 순수 mitochondria는 mitochondrial 구조와 기능, mitochondrial 프로테옴 및 lipidome과 mitochondrial DNA의 분석에도 사용?...

purification of mitochondria from yeast cells

Mitochondria ATP 생산의 주요 사이트는 진핵세포 이외의 광합성 세포에 산소 신진 대사 중에 아르<sup&gt; 1</sup&gt;. 이러한 복잡한 organelles은 apoptotic 세포의 죽음에 필수적인 역할을<sup&gt; 2</sup&gt;, 세포 생존<sup&gt; 3</sup&gt; 포유 동물 개발<sup&gt; 4</sup&gt;의 연결을 개발 및 기능<sup&gt; 4</sup&gt;, 세포내 신호<sup&gt; 5</sup&gt;과 장수 규제<sup&gt; 6</sup&gt;. mitochondria에 의해 제어되는 이러한 복잡한 생물 학적 과정에 대한 우리의 이해는 형태, 그들의 단백질과 지질 조성, 그들의 DNA의 무결성, 그들의 수많은 중요한 기능을 평가하기위한 강력한 방법에 의존합니다. 신진 효모<em&gt; Saccharomyces cerevisiae</em&gt; 주석 게놈하고 잘 정의된 프로테옴과 유전자와 화학적으로 manipulable 단세포 진핵세포 생물은 mitochondria의 필수적인 생물 학적 기능을 기본 분자 및 세포 메커니즘을 연구를위한 가치있는 모델입니다. 연구 이러한 유형의 경우, 그것은 매우 순수한 mitochondria을 가지고 중요합니다. 여기 mitochondria 효모의 정화에 대한 신속하고 효과적인 방법에 대한 자세한 설명을 제시한다. 이 방법은 다른 organelles에 의해 오염의 기본적 자유 및 정화 후 자신의 구조와 기능 무결성을 유지 높은 순수 mitochondria의 절연을 수 있습니다. 이 방법에 의해 정화 Mitochondria은 필수 mitochondrial 프로세스의 셀 - 무료 reconstitution에 적합하고 mitochondrial 구조와 기능, mitochondrial 프로테옴 및 lipidome하고, mitochondrial DNA의 분석에 사용될 수 있습니다.

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

Purification of Mitochondria from Yeast Cells

우리는 효모에서 mitochondria의 정화에 대한 신속하고 효과적인 방법을 설명<em&gt; Saccharomyces cerevisiae</em&gt;. 이 방법은 다른 organelles에 의해 오염의 기본적 자유 및 정화 후 자신의 구조와 기능 무결성을 유지 순수 mitochondria의 높은 수율 격리 수 있습니다.

효모 세포 Mitochondria의 정화

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 조회수.  Concordia University. 우리는 효모에서 mitochondria의 정화에 대한 신속하고 효과적인 방법을 설명 Saccharomyces cerevisiae. 이 방법은 다른 organelles에 의해 오염의 기본적 자유 및 정화 후 자신의 구조와 기능 무결성을 유지 순수 mitochondria의 높은 수율 격리 수 있습니다.

비디오: 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.

CD4의 분리 + Miltenyi 맥을 정제를 사용하여 마우스 림프절에서 T 세포

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

연구

생물학

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.

유기 용매 및 초산없이 쿠매시 G - 250로 젤의 단백질 염색법

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.

골격근에서 Progenitors의 정화

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.

높은 활성 효모 변이의 색층 정화

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.

단일 미토콘드리아 과산화물 온전한 마음에 점멸 또는 공 촛점 이미징<em&gt; 생체</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.

현지화 번역의 메커니즘을 연구하는 효모 미토콘드리아와의 mRNA 관련의 분리

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.

조직 해리 차동 여과 이식 미토콘드리아의 신속한 분리 및 정제

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.

분리 및 배양 세포 및 마우스 조직에서 미토콘드리아의 기능적 분석

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.

진핵 세포에서 염색체 외 원형 DNA의 게놈 차원의 정화

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.

마우스 혈액에서 혈소판의 정제

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