Name: isolation of cellular lipid droplets: two purification techniques starting from yeast cells and human placentas
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이 작품은 PD에 PD로 미국 심장 협회 (American Heart Association) 상 13SDG14500046, 지속 가능한 에너지 교육 및 연구 센터 상 (테네시 Univ.의)에 의해 지원되며, JM에 대한 의사의 의학 교육 및 연구 재단 (테네시 Univ.의)에 의해 수상했다 그의 떨고 인큐베이터, 탁상 원심 분리기, 웨스턴 블롯 분석 장비의 사용 에릭 T. Boder (테네시 Univ.의), 저자는 spheroplasts에 분열 효모를 변환하는 프로토콜 캐롤라인 Leplante (. 예일 대학교를) 감사를위한 센터 자신의 초 원심 분리기의 사용 환경 생명 공학 명 (대학 테네시), 효모 항체 귄터 다음 세대 (그라츠 대학 기술, 오스트리아.) 기술 지원 산부인과 명 (대학 테네시 의료 센터)의 부서의 직원 .

1. (분열) 효모 세포에서 지질 방울을 분리 효모 사카로 마이 세스 세레 비시 애의 신진 인기 모델 생물에서 물방울의 분리는 다음 프로토콜 6과 거의 동일합니다. 준비의 차이가 설명되어 있습니다. 1. 성장 효모 세포 <ol><li> 미디어를 준비합니다. 유리 병 또는 배양 플라스크에 dH보다 2 O 리터당 YE5S 분말 36g을 결합합니다. 미디어의 ...

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Chem. 287, 2273-2279 (2012).</li></ol>

<html> 이 프로토콜 내에서 중요한 단계 배양 세포의 성장하는 동안 미디어 및 세포 밀도와 일치해야합니다. 세포 지질 방울들이 관련된 단백질이 세포가 배양 된 17되는 환경에 크게 의존한다는 점에서 독특합니다. 따라서, 세포가 성장되어있는 미디어와 세포의 밀도는 밀접 용해 전에 모니터링해야한다. 지질 방울의 단?...

세포의 지질 방울은 세포의 여러 기능을 제공 동적 세포 기관이다. 이들은 에너지로 변환하거나 인지질 합성을 위해 사용될 수있다 중성 지질, 스토리지 허브이다. 방울은 동맥 경화증, 비만과 관련된 대사 질환 등의 생리 학적 및 병리학 적 조건에서 중심 역할을하고, 또한 전염병 1,2. 또한, 그들은 바이오 디젤 연료에 대한 흥미로운 소스입니다. 지질 방울의 세포 역할에 대한 많은 정보가 광범위한 생물 3에서 정제 방울의 프로테옴 lipidomic 분석에서 얻을 수있다. 이 생명체는 박테리아 4,5, 효모 6-11 포함 12,13 식물, 14 선충, 그리고 15, 16 비행했다. 인간 대사 질환에서 지질 소적의 역할에 관심을 감안할 때, 액적 또한 배양 된 동물 세포에서 분리 된nimal 조직. 배양 세포주는 3T3-L1 지방 세포 (17), 중국 햄스터 난소 (CHO) 세포 K2 18 인간 hepatocyes 19,20 및 상피 세포 라인 (21)을 포함했다. 물방울이 고립되어있는 동물 조직 마우스 골격근 22 일 간 (23) 및 유 방 땀 샘 (23)를 포함했다. 전술 한 바와 같이, 대부분의 액적 분리 연구의 목표는 결합 계수 및 중립 및 인지질에 lipidomic 분석 프로테옴 분석을 수행하는 것이다. 중성 지질시기 - 지질 방울의 대부분의 수많은 구성 요소 - 대부분의 다른 세포 물질보다 밀도이며, 액적 분리 전통적 밀도 구배 원심 분리를 사용하여 수행되었다. 그 기술은 여기에 제시된 두 수험 공부의 중심이다. 이전 기술 6,24은 배양 분열 효모 세포에서 물방울의 분리의 시각적 표현으로 결합 및 수정및 noncultured 인간의 세포는 태반 조직에서 얻을. 목표는 액적 분리를위한 시작점으로서이 크게 다른 세포 유형을 선택하여이 기술의 광범위한 응용을 보여주는 것이다. 이 기술은 대부분의 생물에서 물방울을 분리하고자하는 사람들에게 유용하게 쓰일 수있을 것이다. 프로토콜 하나는 진핵 세포 분열 (25) 중에 액적 형성을 관찰하기위한 모델로서 이용되어왔다 분열 효모 Schizosaccharomyces 용의 pombe로부터 지질 방울의 분리를 설명한다. 신진 효모 사카로 마이 세스 세레 비시 애 지질 방울 생물학 연구의 모델 생물로 광범위하게 사용되어왔다. 프로토콜 1 생물과 준비의 차이 모두에 적용이 강조된다. 프로토콜 2는 인간의 장기 태반에서 얻을 수 차례에 태반 융모 세포에서 지질 방울의 분리에 대해 설명합니다.용어 태반의 수집은 안전하고 윤리적으로 지질 방울의 상당한 숫자가 포함 쉽게 사용할 수 인체 조직 26, 200-250 g을 수득 할 수있는 독특한 기회를 제공합니다. 이것은 물방울이 배양 된 세포에서 발생한 높은 진핵 가장 지질 액적 분리 작업 대조적이다. 그 연구에서, 지방산은 종종 중성 지질 따라서 방울의 성장의 합성을 촉진하는 문화에 추가됩니다. 이 지질 방울는 태반 조직의 기본 조건이 형성되어 여기에 작업 대조적이다. 지질 방울 분획의 순도는 소기관 마커 항체를 사용하여 웨스턴 블롯 분석에 의해 결정된다. 이 두 프로토콜은 다음, 프로테옴 lipidomic 분석에 적합한 지질 방울 분수를 얻을 것입니다.

<table border="0" cellpadding="0" cellspacing="0" width="802">
	<colgroup>
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">PROTOCOL #1:&#160;</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">1.Growing yeast cells and converting to spheroplasts</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Edinburgh Minimal Media (EMM)</td>
			<td>Sunrise Science Products</td>
			<td style="width:119px;">2005</td>
			<td></td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;">Yeast extract with 5 supplements (YE5S)</td>
			<td>Sunrise Science Products</td>
			<td style="width:119px;">2011</td>
			<td style="width:167px;">YE5S media with 225 mg/ml of each supplement: adeninie, histidine, leucine, lysine, uracil. The equivalent for budding yeast would be YPD.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">YPD powder</td>
			<td>Sunrise Science Products</td>
			<td style="width:119px;">1875</td>
			<td>For S. cerevisiae&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sorbitol</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP439</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Lytic Enzyme</td>
			<td>MP Biomedicals</td>
			<td style="width:119px;">215352610</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lysing Enzymes from Trichoderma harzianum</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">L1412</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zymolayse-20T</td>
			<td>Sunrise Science Products</td>
			<td style="width:119px;">N0766391</td>
			<td>For S. cerevisiae&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BODIPY 493/503</td>
			<td>Invitrogen</td>
			<td style="width:119px;">D-3922</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">12-544-7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope Cover Glass</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">12-542-B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plastic transfer pipette</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">137115AM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 liter glass bottle</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">250 ml flask</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2.8 liter flasks</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2. Yeast lipid droplet isolation</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCl</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ficoll 400</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12-14k Spectra/Por Dialysis Membrane</td>
			<td>SpectrumLabs</td>
			<td style="width:119px;">132680</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA-free Protease Inhibitor Cocktail Tablets</td>
			<td>Roche Diagnostics</td>
			<td style="width:119px;">11873580001</td>
			<td>irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dounce Homogenizer&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">D9938</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultracentrifuge Tubes 25x89mm (for SW28)</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;">355642</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12-14k Spectra/Por Dialysis Membrane</td>
			<td>SpectrumLabs</td>
			<td style="width:119px;">132680</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Name of Equipment</td>
			<td style="width:163px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Temperature-controlled shaker</td>
			<td>New Brunswick Scientific</td>
			<td style="width:119px;">C25KC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermo Sorvall Legend XTR centrifuge</td>
			<td>Thermo-Scientific</td>
			<td style="width:119px;">75004521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Swinging Bucket Centrifuge Rotor</td>
			<td>Thermo-Scientific</td>
			<td style="width:119px;">75003607</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fiberlite* F15-6x100y Fixed-Angle Rotor</td>
			<td>Thermo-Scientific</td>
			<td style="width:119px;">75003698</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultracentrifuge LB-M</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SW28 Ultracentrifuge Rotor</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;">342204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PROTOCOL #2&#160;</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1. Placental villous cells isolation</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Disposable underpads</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">23666062</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Autoclavable pan (container), 3L</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">1336110</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fine scissors, sharp-sharp, straight</td>
			<td>Fine science tools</td>
			<td style="width:119px;">1406011</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">London Forceps</td>
			<td>Fine science tools</td>
			<td style="width:119px;">1108002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dumont #7b Forceps</td>
			<td>Fine science tools</td>
			<td style="width:119px;">1127020</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Razor blades</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S65921</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Screen cup for CD-1</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S1145</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">40 mesh screen&#160;</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S0770</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fisherbrand cell stainers 100&#956;m</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">22363549</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">150 mm Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">NC9054771</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S642</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">P333</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KH2PO4</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">P386</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S374</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-glucose</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">D16</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP310</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2.5% trypsin 10x</td>
			<td>Invitrogen</td>
			<td style="width:119px;">15090046</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNase I grade II, from bovine pancreas</td>
			<td>Roche Applied Science</td>
			<td style="width:119px;">10104159001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium bicarbonate solution</td>
			<td>Sigma-aldrich</td>
			<td style="width:119px;">S8761</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">500 ml Erlenmeyer flasks</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">250 ml beakers</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">15 ml centrifuge tubes</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">10 ml serological pipettes</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">50 ml centrifuge tubes</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">DMEM</td>
			<td style="width:163px;">Invitrogen</td>
			<td style="width:119px;">11965084</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2. Lipid droplets isolation from villous placental cells</td>
			<td></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCl</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-Sucrose</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Carbonate&#160;</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP357</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA-free protease inhibitor cocktail tablets</td>
			<td>Roche Diagnostics</td>
			<td style="width:119px;">11873580001</td>
			<td>irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dounce homogenizer&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">D9938</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultracentrifuge tubes 25x89mm (for SW28)</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;">355642</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra-Clear centrifuge tubes 14x89mm (for SW41)</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;">344059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable borosilicate glass pasteur pipets</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">1367820C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Name of Equipment</td>
			<td style="width:163px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Biological safety hood&#160;</td>
			<td style="width:163px;">Thermo-Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Waterbath</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Temperature-controlled shaker</td>
			<td>New Brunswick Scientific</td>
			<td style="width:119px;">C25KC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermo Sorvall Legend XTR centrifuge</td>
			<td>Thermo-Scientific</td>
			<td style="width:119px;">75004521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Swinging Bucket Centrifuge Rotor</td>
			<td>Thermo-Scientific</td>
			<td style="width:119px;">75003607</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultracentrifuge LB-M</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SW28 Ultracentrifuge Rotor</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;">342204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SW41 Ti Ultracentrifuge Rotor</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;">331336</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Western blot</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">IRDye 680 Goat Anti-Rabbit IgG</td>
			<td style="width:163px;">LI-COR</td>
			<td style="width:119px;">926-68071</td>
			<td>dilution 1:15000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">IRDye&#160; 800CW Goat Anti-Mouse IgG</td>
			<td style="width:163px;">LI-COR</td>
			<td style="width:119px;">926-32210</td>
			<td>dilution 1:5000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">NuPAGE&#174; Novex&#174; 12% Bis-Tris gels</td>
			<td style="width:163px;">Invitrogen</td>
			<td style="width:119px;">NP0341</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">primary antibodies for PROTOCOL #1</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:353px;">Erg6p</td>
			<td style="width:163px;">gift from Dr. G. Daum</td>
			<td style="width:119px;">Graz University of Technology, Austria</td>
			<td>dilution 1:5000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Dpm1p</td>
			<td style="width:163px;">Abcam</td>
			<td style="width:119px;">ab113686</td>
			<td>4 &#956;g/ml</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:353px;">Por1p</td>
			<td style="width:163px;">gift from Dr. G. Daum</td>
			<td style="width:119px;">Graz University of Technology, Austria</td>
			<td>dilution 1:5000</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:353px;">Pma1p</td>
			<td style="width:163px;">gift from Dr. G. Daum</td>
			<td style="width:119px;">Graz University of Technology, Austria</td>
			<td>dilution 1:10000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Vma1p (anti-ATP6V1A)</td>
			<td style="width:163px;">Abcam</td>
			<td style="width:119px;">ab113745</td>
			<td>0.5 &#956;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;"></td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">primary antibodies for PROTOCOL #2</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">perilipin 2 (anti-ADFP)</td>
			<td style="width:163px;">Abcam</td>
			<td style="width:119px;">ab52355</td>
			<td>2 &#956;g/ml</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:353px;">calnexin</td>
			<td style="width:163px;">Cell Signaling technology</td>
			<td style="width:119px;">2679</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">GM130</td>
			<td style="width:163px;">Biorbyt</td>
			<td style="width:119px;">orb40533</td>
			<td>dilution 1:25</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:353px;">COX IV</td>
			<td style="width:163px;">Cell Signaling technology</td>
			<td style="width:119px;">4850</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">MEK1</td>
			<td style="width:163px;">Biorbyt</td>
			<td style="width:119px;">orb38775</td>
			<td>dilution 1:50</td>
		</tr>
	</tbody>
</table>

isolation of cellular lipid droplets: two purification techniques starting from yeast cells and human placentas

지질 방울은 대부분의 진핵 및 원핵 특정 세포에서 발견 될 수있는 동적 소기관이다. 구조적으로, 방울 인지질 단층으로 둘러싸인 중성 지질의 핵심으로 구성되어 있습니다. 액적 셀룰러 역할을 결정하는데 가장 유용한 기술 중 하나는 작은 방울과 함께 단리 할 수​​있다 결합 단백질의 프로테옴 식별되었다. 분열 효모 및 인간의 태반 융모 세포 : 여기, 두 가지 방법은 두 개의 광범위한 진핵 생물에서 지질 방울과 그들의 결합 된 단백질을 분리 할 수​​ 있도록 서술되어있다. 두 기술의 차이가 있지만, 주요 방법 - 밀도 구배 원심 분리 - 모두 준비가 공유됩니다. 이 제시 액적 분리 기법의 넓은 적용 가능성을 보여준다. 첫 번째 프로토콜에서 효모 세포는 세포벽의 소화 효소에 의해 spheroplasts로 변환됩니다. 그 결과 spheroplasts는 신사된다LY는 헐렁한 균질화에 용해. 피콜은 밀도 구배를 제공하기 위해 파쇄물을 첨가하고, 혼합물을 세 번 원심 분리된다. 첫번째 스핀 후, 지질 방울을 소포체 (ER), 세포막과 액포 함께 원심 분리기 튜브의 하얀색 부동 층에 지역화된다. 두 개의 연속 스핀은 다른 세 가지 세포 기관을 제거하는 데 사용됩니다. 결과는 작은 방울과 결합 단백질을 가지고 층이다. 두 번째 프로토콜에서, 태반 융모 세포는 트립신과 DNA 분해 효소 나 세포가 느슨한 맞는 균질의 균질화되어있는 소화 효소에 의해 인간의 장기 태반에서 격리됩니다. 저속 및 중속 원심 분리 단계는 손상되지 않은 세포, 세포 파편, 핵, 미토콘드리아를 제거하는 데 사용됩니다. 자당은 밀도 구배를 제공하기 위해 파쇄 액에 첨가하고, 혼합물을 다른 cellula로부터 지질 방울을 분리하기 위해 원심 분리R 분수. 두 프로토콜의 지질 방울의 순도는 웨스턴 블롯 분석에 의해 확인된다. 모두 최상의 상태로 준비에서 물방울 분수 이후 프로테오믹스 및 lipidomic 분석에 적합하다.

예상대로 밀도 구배 원심 분리에 근무하는 경우, 부동 층은 지질 방울을 포함해야하고, 고속 스핀의 진행 내내 다른 소기관 고갈 될. 프로토콜 1의 경우, 웨스턴 블롯 마커 지질 방울 (Erg6p)에 대한 항체, 효모, ER (Dpm1p), 미토콘드리아 (Por1p)의 지질 방울과 상호 작용하는 것으로 밝혀졌다 세포 기관으로 수행하고, 세포막 (Pma1p), 및 액포 (Vma1p). 각 3 스핀의 ?...

Watch this Scientific Journal Video about Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas at JoVE.com

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

1)에서 세포 지질 방울을 분리 효모 세포, 2) 인간의 태반이 제시하는 두 가지 기술. 두 절차의 핵심은 방울을 함유하는 생성 된 부동 층은 쉽게 눈에 의해 시각을 추출하고, 순도에 대한 웨스턴 블롯 분석에 의해 정량화 될 수있는 밀도 구배 원심 분리된다.

세포 지질 방울의 분리 : 효모 세포와 인간의 태반에서 시작 두 정화 기술

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of ...

Research

JoVE Journal

Bioengineering

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

인간 세포 배양을위한 회전 세포 배양 시스템 : 모델로 인간 Trophoblast 셀

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

연구

생체공학

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

타겟 안정 동위 원소 희석 액체 크로마 토그래피 - 질량 분광법 분석을위한 세포 지질 추출

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

개발, 확대,<em&gt; 생체 내</em&gt; 인간 배아 줄기 세포 (hESCs에)에서 인간 NK 세포의 모니터링 및 및 유도 된 pluripotent 줄기 세포 (유도 만능)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

신경 조직의 재생을위한 가용성 및 불용성 바이오틴 단백질의 발현, 분리 및 정제

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

미세 유체 제트를 사용하여 지질 이중층 소포 세대

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

수술 조직에서 기본 인간 대장 종양 세포의 분리 및 견인 세포 계측법 부드러운 탄성 기판에 직접 배양

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol

에탄올에 리그 노 셀룰로즈의 생물에 대한 강력한 펜 토스 발효 효모의 진화를위한 기술

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

인간의 치경과 파생 셀 Osteogenic 활동에 비타민 D의 효과에서 중간 엽 줄기 세포의 분리

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

자동화된 미생물체 시스템을 사용하여 중국 햄스터 난소 세포의 단일 클론 항체의 정화 및 분석

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Falcarindiol 인간 중간 엽 줄기 세포에 의해 포함 된 새로운 지질 코팅 나노 입자의

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...