Name: cell fractionation of u937 cells in the absence of high-speed centrifugation
Uploaded: 2019-01-18T15:00:00
Description: Watch this Scientific Journal Video about Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation at JoVE.com

nih-1r15hl13565-01 为 timothy j. lrocca 提供了支持

1. 准备缓冲器和试剂
注: 请参见表 1。
<ol><li>准备缓冲液 a、裂解缓冲液 b、样品缓冲液和数字宁的溶液。<ol>
<li>加入 8.77 g 的氯化钠和50毫升的 hepes (1 m, ph 7.4) 到900毫升去离子水, 用去离子水将最终体积调整为1升。 注: 最终浓度为 150 mm 氯化钠和 50 mm hepes。</li>
		<li>加入20毫升的 hepes (1 m, ph 7.4) 制备裂解缓冲液 b, kcl 0.75 g, mgcl2, 2 ml 乙二胺四乙酸 (0.5 m edta...

<ol>
	<li>Baghirova, S., Hughes, B. G., Hendzel, M. J., Schulz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+fractionation+and+isolation+of+subcellular+proteins+from+tissue+or+cultured+cells.">Sequential fractionation and isolation of subcellular proteins from tissue or cultured cells.</a> MethodsX. 2, e440-e445 (2015).</li><li>Hwang, S., Han, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+fractionation+for+identification+of+biomarkers:+Serial+detergent+extraction+by+subcellular+accessibility+and+solubility.">Subcellular fractionation for identification of biomarkers: Serial detergent extraction by subcellular accessibility and solubility.</a> Methods in Molecular Biology. 1002, 25-35 (2013).</li><li>Song, Y., Hao, Y., 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=Sample+preparation+project+for+the+subcellular+proteome+of+mouse+liver.">Sample preparation project for the subcellular proteome of mouse liver.</a> Proteomics. 6 (19), 5269-5277 (2006).</li><li>Lenstra, J. A., Bloemendal, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topography+of+the+total+protein+population+from+cultured+cells+upon+fractionation+by+chemical+extractions.">Topography of the total protein population from cultured cells upon fractionation by chemical extractions.</a> European Journal of Biochemistry. 135 (3), 413-423 (1983).</li><li>Michelsen, U., von Hagen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+19+Isolation+of+Subcellular+Organelles+and+Structures.">Chapter 19 Isolation of Subcellular Organelles and Structures.</a> Methods in Enzymology. 463 (C), 305-328 (2009).</li><li>Stimpson, S. E., Coorssen, J. R., Myers, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+isolation+of+mitochondria+for+proteomic+analyses).">Optimal isolation of mitochondria for proteomic analyses).</a> Analytical Biochemistry. 475, 1-3 (2015).</li><li>Williamson, C. D., Wong, D. S., Bozidis, P., Zhang, A., Colberg-Poley, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Endoplasmic+Reticulum,+Mitochondria,+and+Mitochondria-Associated+Membrane+and+Detergent+Resistant+Membrane+Fractions+from+Transfected+Cells+and+from+Human+Cytomegalovirus-Infected+Primary+Fibroblasts.">Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane and Detergent Resistant Membrane Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts.</a> Current protocols in cell biology. 68, (2015).</li><li>Sundstr&#246;m, C., Nilsson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+a+human+histiocytic+lymphoma+cell+line+(U-937).">Establishment and characterization of a human histiocytic lymphoma cell line (U-937).</a> International Journal of Cancer. 17 (5), 565-577 (1976).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Proceedings of the National Academy of Sciences. 76 (9), 4350-4354 (1979).</li><li>Barber, R. D., Harmer, D. W., Coleman, R. A., Clark, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GAPDH+as+a+housekeeping+gene:+analysis+of+GAPDH+mRNA+expression+in+a+panel+of+72+human+tissues.">GAPDH as a housekeeping gene: analysis of GAPDH mRNA expression in a panel of 72 human tissues.</a> Physiological Genomics. 21 (3), 389-395 (2005).</li><li>Hodge, T., Colombini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+metabolite+flux+through+voltage-gating+of+VDAC+channels.">Regulation of metabolite flux through voltage-gating of VDAC channels.</a> Journal of Membrane Biology. 157 (3), 271-279 (1997).</li><li>Therien, a. G., Blostein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+sodium+pump+regulation.+American+journal+of+physiology.">Mechanisms of sodium pump regulation. American journal of physiology.</a> Cell physiology. 279 (3), C541-C566 (2000).</li><li>Devarajan, P., Stabach, P. R., De Matteis, M. A., Morrow, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Na,K-ATPase+transport+from+endoplasmic+reticulum+to+Golgi+requires+the+Golgi+spectrin-ankyrin+G119+skeleton+in+Madin+Darby+canine+kidney+cells.">Na,K-ATPase transport from endoplasmic reticulum to Golgi requires the Golgi spectrin-ankyrin G119 skeleton in Madin Darby canine kidney cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 94 (20), 10711-10716 (1997).</li><li>McCaig, W. D., Patel, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperglycemia+potentiates+a+shift+from+apoptosis+to+RIP1-dependent+necroptosis.">Hyperglycemia potentiates a shift from apoptosis to RIP1-dependent necroptosis.</a> Cell Death Discovery. 4, 55 (2018).</li><li>Simpson, 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=Homogenization+of+Mammalian+Tissue.">Homogenization of Mammalian Tissue.</a> Cold Spring Harbor Protocols. 2010 (7), (2010).</li></ol>

该方案的发展源于无法分离线粒体和膜样本, 使用商业上可获得的试剂盒, 以分析在坏死14期间的蛋白质定位.预制试剂盒的主要局限性是, 它们无法适应个别研究人员的需要, 每个样本的费用有限, 能够加工的样品数量有限。这里介绍的方法可以在不使用昂贵的试剂的情况下进行, 也不需要昂贵的设备。这种方法可以进行缩放, 以容纳任意数量的细胞, 并能够进行修改, 以适应研究人?...

在检查真核细胞中的分子途径时, 通常需要可靠地识别蛋白质定位。研究人员利用获得亚细胞组分的方法来更仔细地检查感兴趣的细胞成分。
大多数现有的细胞分馏方法一般分为两大类, 即基于洗涤剂的 1、2和基于超离心的3、4、5, 它们可以是以速度、精度和成本为区别。基于洗涤剂的协议依赖于使用具有不断增加的洗涤剂强度的缓冲液来溶解细胞的不同成分。这是一种快速方便的样品处理方法, 如果样品数量和大小较小, 则具有成本效益。以洗涤剂为基础的试剂盒可以从细胞中分离细胞质、膜细胞器 (混合组分) 和核组分。然而, 与这些试剂盒相关的一些缺点限制了他们对研究人员的有用性。它们旨在轻松分离单元的一个或两个组件, 但无法同时从样本中分离所有分数。洗涤剂的使用意味着质膜和膜封闭的细胞器将同样溶解, 因此, 无法相互分离。另一个复杂因素来自于这些试剂盒中的专有组件, 这些组件阻止研究人员更改特定应用的条件。最后, 它们的用途有限, 对于规模较大的实验来说可能费用高得令人望而却步。非洗涤剂为基础的试剂盒存在于线粒体的分离, 但是, 它们不是用来分离质膜的, 样品的收率明显低于密度离心分离协议6,7.
利用超离心获得分数的方法比基于洗涤剂的试剂盒更耗时, 但往往会产生更纯净的组分。要在不首先溶解细胞的情况下从细胞中分离等离子体膜 (导致膜细胞器污染), 需要用非洗涤剂方法对其进行裂解, 然后通过差异分离细胞成分离心--质膜分离需要 100, 000xg 的速度才能完成。在许多情况下, 差动离心之后必须进行等温密度梯度离心, 以进一步分离细胞组分或去除污染物。虽然这些方法是彻底和可修改的, 缺点包括成本, 时间消耗, 以及需要一个超离心分离的分数和进一步纯化通过密度梯度离心。大多数高速离心机的费用对个别调查人员来说是令人望而却步的, 而且往往是学术机构共享的核心设备。因此, 在这些情况下, 超离心机的可用性变得令人望而却步。
在这个分馏协议中, 我们证明了在不使用溶解性洗涤剂和不高速离心的情况下分离亚细胞分数。这种方法将使研究人员能够分离真核细胞的质膜、线粒体和细胞质成分, 并将分数之间的污染降至最低。

<table>
	<tbody>
		<tr>
			<td>Digitonin</td>
			<td>TCI Chemicals</td>
			<td>D0540</td>
			<td>For Cytoplasm Extraction</td>
		</tr>
		<tr>
			<td>D-Mannitol</td>
			<td>Sigma-Aldrich</td>
			<td>M4125</td>
			<td>For Lysis buffer B</td>
		</tr>
		<tr>
			<td>Dounce homogenizer</td>
			<td>VWR</td>
			<td>22877-282</td>
			<td>For Homogenization</td>
		</tr>
		<tr>
			<td>end-over-end rotator</td>
			<td>Barnstead</td>
			<td>N/A</td>
			<td>For Cytoplasm Extraction</td>
		</tr>
		<tr>
			<td>ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA)</td>
			<td>Alfa Aesar</td>
			<td>J61721</td>
			<td>For Lysis buffer B</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E7889</td>
			<td>For Lysis buffer B</td>
		</tr>
		<tr>
			<td>GAPDH (14C10)</td>
			<td>Cell Signalling Technologies</td>
			<td>2118</td>
			<td>For detection of cytoplasmic fractions on western blot, dilution: 1:10000</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>VWR</td>
			<td>J848</td>
			<td>For Lysis buffers A and B</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td>For Lysis buffer B</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Alfa Aesar</td>
			<td>12315</td>
			<td>For Lysis buffer B</td>
		</tr>
		<tr>
			<td>Na, K-ATPase a1 (D4Y7E)</td>
			<td>Cell Signalling Technologies</td>
			<td>23565</td>
			<td>For detection of plasma membrane fractions on western blot, dilution: 1:1000</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>793566</td>
			<td>For Lysis buffer A</td>
		</tr>
		<tr>
			<td>phenylmethanesulfonyl fluoride (PMSF)</td>
			<td>VWR</td>
			<td>M145</td>
			<td>For Cytoplasm Extraction and Homogenization Buffer</td>
		</tr>
		<tr>
			<td>probe sonicator</td>
			<td>Qsonica</td>
			<td>Q125-110</td>
			<td>For Final Samples</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail, General Use</td>
			<td>VWR</td>
			<td>M221-1ML</td>
			<td>For Cytoplasm Extraction</td>
		</tr>
		<tr>
			<td>refrigerated centrifuge</td>
			<td>Beckman-Coulter</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>VWR</td>
			<td>227</td>
			<td>For Sample buffer</td>
		</tr>
		<tr>
			<td>sodium orthovanadate (SOV)</td>
			<td>Sigma-Aldrich</td>
			<td>450243</td>
			<td>For Lysis buffers A and B</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td>For Lysis buffer B</td>
		</tr>
		<tr>
			<td>Tris-buffered Saline (TBS)</td>
			<td>VWR</td>
			<td>788</td>
			<td>For Sample buffer</td>
		</tr>
		<tr>
			<td>VDAC (D73D12)</td>
			<td>Cell Signalling Technologies</td>
			<td>4661</td>
			<td>For detection of mitochondrial fractions on western blot, dilution: 1:1000</td>
		</tr>
	</tbody>
</table>

cell fractionation of u937 cells in the absence of high-speed centrifugation

在该协议中, 我们详细介绍了在不使用超离心或不滥用洗涤剂的情况下获取 u937 细胞亚细胞分数的方法。该方法利用低渗缓冲液、数字素、机械裂解和差动离心分离细胞质、线粒体和质膜。这个过程可以扩大规模, 以满足研究人员的需求, 既便宜又直接。这种方法将使研究人员能够在没有专门离心机和商业试剂盒的情况下确定细胞中蛋白质的定位, 这两种方法的成本都高得令人望而却步。我们已经成功地使用这种方法分离人单细胞系 u937 中的细胞质、质膜和线粒体蛋白。

利用上面详细的协议和图 1所示的协议, 成功地完成了悬浮液中生长的未分化 u937 8 细胞的成功分馏。用这种方法获得的样品采用湿法转移到聚偏氟乙烯 (pvdf) 膜上进行了西方印迹9 。随后用细胞质、线粒体和膜局部蛋白标记抗体对膜进行了检测 (图 2,图 3,?...

Watch this Scientific Journal Video about Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation at JoVE.com

Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation

高速离心条件下 u937 细胞的细胞分裂

在这里, 我们提出了一个在不使用高速离心的情况下分离 u937 细胞的质膜、细胞质和线粒体的方案。该技术可用于纯化亚细胞组分, 以便随后通过免疫印迹检查蛋白质定位。

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. ...

该协议允许研究人员获得细胞的不同分数，而无需使用专用设备、昂贵的试剂或复杂的技术。该协议的主要优点是技术的简单性，这使得它可修改和易于执行。演示这个程序的将是马特 · 德拉贡、罗布 · 哈卢斯卡和亚历克萨 · 霍奇斯。开始离心，将准备好的细胞悬浮液产生颗粒。拆下上一液，并在室温 PBS 中重新悬浮颗粒。将电池悬浮液以400 xg离心10分钟。然后去除上清液，在冰冷的缓冲液中重新悬浮细胞颗粒。根据文本协议准备缓冲区解决方案。将电池悬浮液在400 xg下离心10分钟，然后取出上一代。将缓冲液中的颗粒重新悬浮 A 溶液，最终浓度为每毫升 2 亿细胞，并轻轻移液器以分解任何团块。在此孵育后，细胞悬浮液在结束旋转器的端端上 4

Research

JoVE Journal

Biochemistry

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

巨囊泡封装微球通过一个水包油乳液的离心制备

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

科研

生物化学

Nitrogen Cavitation and Differential Centrifugation Allows for Monitoring the Distribution of Peripheral Membrane Proteins in Cultured Cells

氮气气蚀和差速离心允许监测外周血膜蛋白在细胞中的分布

Cultured cells are useful for studying the subcellular distribution of proteins, including peripheral membrane proteins. Genetically encoded fluorescently ...

Fractionation for Resolution of Soluble and Insoluble Huntingtin Species

可溶性和不溶性杭丁顿蛋白的分离分解

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

膜分馏和 gtpase-关联免疫吸附法检测小 gtpase 预基化和 gtp 结合

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

利用生物测定引导分馏法鉴定海洋鳗信息素

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

基于原生自荧光的sFLIM测量植物部分荧光探针和木质素之间的相互作用

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

使用免疫增强技术的超高速西布洛特

A western blot (also known as an immunoblot) is a canonical method for biomedical research. It is commonly used to determine the relative size and ...

Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

通过等密度梯度纯化对U937细胞进行细胞分级分离

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and ...

Quantifying the Binding Interactions Between Cu(II) and Peptide Residues in the Presence and Absence of Chromophores

Quantifying the Binding Interactions Between CuII and Peptide Residues in the Presence and Absence of Chromophores

Copper(II) is an essential metal in biological systems, conferring unique chemical properties to the biomolecules with which it interacts. It has been ...