Name: nitrogen cavitation and differential centrifugation allows for monitoring the distribution of peripheral membrane proteins in cultured cells
Uploaded: 2017-08-18T14:00:00
Description: Watch this Scientific Journal Video about Nitrogen Cavitation and Differential Centrifugation Allows for Monitoring the Distribution of Peripheral Membrane Proteins in Cultured Cells at JoVE.com

这项工作是由 GM055279、 CA116034 和 CA163489 资助的。

1.缓冲区和设备准备工作 <ol><li>冷藏 45 毫升细胞破坏炸弹、 15 毫升管和离心管在 4 ° C.</li>
	<li>准备放松 25 毫升的同质化缓冲区每 4 ° C.添加一个蛋白酶抑制剂平板电脑只是在使用前 2 x 10 7 细胞。 
	注： 同质化缓冲区通常包含氯化钾，而不是氯化钠，更好地反映细胞内盐组成。在本议定书中所使用的同质化缓冲区包括 10 毫米复在 pH 7.4，10 毫米 KCl 和 1.5 毫米氯化镁 2 （以下...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preperation+of+plasma+membrane+vesicles+by+nitrogen+cavitation.">Preperation of plasma membrane vesicles by nitrogen cavitation.</a> TCA manual / Tissue Culture Association. 3 (3), 653-654 (1977).</li><li>Brock, T. G., Paine, R., Peters-Golden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+5-lipoxygenase+to+the+nucleus+of+unstimulated+rat+basophilic+leukemia+cells.">Localization of 5-lipoxygenase to the nucleus of unstimulated rat basophilic leukemia cells.</a> J Biol Chem. 269 (35), 22059-22066 (1994).</li><li>Dowben, R. M., Gaffey, A., Lynch, P. 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+liver+and+muscle+polyribosomes+in+high+yield+after+cell+disruption+by+nitrogen+cavitation.">Isolation of liver and muscle polyribosomes in high yield after cell disruption by nitrogen cavitation.</a> FEBS Letters. 2 (1), (1968).</li><li>Dai, Q., 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=Mammalian+prenylcysteine+carboxyl+methyltransferase+is+in+the+endoplasmic+reticulum.">Mammalian prenylcysteine carboxyl methyltransferase is in the endoplasmic reticulum.</a> J Biol Chem. 273 (24), 15030-15034 (1998).</li><li>Wynne, J. 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空化氮比其他方法的机械破碎的优势是多方面的。也许最显著的好处是它能够有效地还轻轻地同质化标本。而不是生成局部加热伤害减压冷却样品的物理原理像超声和摩擦、 剪切的基础技术。空化也是效率极高，破坏细胞膜的。因为泡沫生成后减压、 气蚀过程的各个单元格内的氮是有限较少的单元格的大小，样本大小或样品浓度。因此，它提供基于间隙的 Dounce 或针通过同质化的额外优势。氮空...

细胞蛋白可以分为两类： 那些与膜和那些不相关联。非膜相关的蛋白位于细胞质、 核质和腔的内质网 (ER) 等细胞器。有膜相关蛋白、 积分和外周两类。积分的膜蛋白也被称为是跨膜蛋白，因为一个或多个段多肽链跨越膜，通常作为疏水性氨基酸组成的 α 螺旋。跨膜蛋白 co-translationally 插入到其生物合成的过程中膜和保持如此配置，直到他们被异化。外周膜蛋白为辅赶入膜，通常由于翻译后修饰与疏水性的分子，如脂类。与内在膜蛋白外周膜蛋白与细胞膜的关联是可逆的可以调节。许多外周膜蛋白功能的信号通路和调节的协会与膜是一种机制激活或抑制通路。这是一个外周膜蛋白的信号分子的一个例子是小蛋白，RAS。经过一系列的修饰，包括修改和金合欢脂类，成熟的 RAS 蛋白改性的 C-末端插入细胞膜细胞质单张。具体地说，细胞膜是 RAS 在那里从事其下游效应器 RAF1。为了防止丝裂素活化蛋白激酶 (MAPK) 信号转导通路的组成性激活，多层次的 RAS 控制到位。除了呈现 RAS GTP 水解成 GDP 处于非活动状态，积极 RAS 也可以释放从细胞膜通过修改或增溶因素抑制信号的相互作用。虽然荧光实时成像提供细胞生物学家观察荧光蛋白标记的外周膜蛋白1的亚细胞定位的机会，仍然迫切需要评价膜协会半定量与简单的生化途径的内源性蛋白。
对蛋白质膜和可溶性组分之间的分配适当的生化评定是取决于两个因素： 细胞同质化和高效分离膜和可溶。虽然一些协议，包括使用最广泛的商业化的套件，取决于洗涤剂基于细胞同质化，这些方法可以通过膜蛋白提取到可溶性阶段2混淆分析。因此，细胞破碎非洗涤剂为主，机械方法提供更清洁的结果。有几种方法的机械破坏的细胞在培养基中培养或收获从血液或器官。这些包括 Dounce 均匀化、 细针中断、 滚珠轴承同质化、 超声和氮空。在这里我们评价氮空，并与其他方法进行比较。氮空化依赖于在高压力下细胞的细胞质中溶解的氮。平衡后, 将细胞悬浮液是突然暴露到大气压力，这样撕开由于迸裂细胞的细胞质中形成氮气气泡。如果压力足够高，氮泡腾可以破坏核3和溶酶体4像细胞器的膜。然而，如果压力保持足够低，减压会破坏细胞膜和 ER 但不是其他细胞器，从而蔓延到指定空化5组织匀浆的细胞质和细胞器完好。为此，氮空化是首选的隔离溶酶体、 线粒体等细胞器方法。
然而，它也是制备匀浆，可以容易地分离成膜和可溶的优秀方法。压力容器 （此后称为"炸弹"） 在空化过程中使用包括承受高压力，与入口的氮气从一辆坦克和可调流量阀出气口交付一个厚不锈钢外壳。
氮空已用于自 20 世纪 60 年代6细胞同质化。1961 年，猎人和 Commerfold7建立氮空作为哺乳动物组织中断的可行选项。自那时以来，科学家已经采用了该技术对各种细胞和组织的成功，和氮空化已成为多个应用程序，包括膜制备8，9，细胞核和细胞器的主食制备10，11和生化活性物质的提取。目前，细胞生物学家更经常雇用其他细胞破碎的方法因为氮同质化的好处不作出广泛的宣传、 氮炸弹是昂贵和有一种误解，相对大量的细胞是必填。氮空化实现无细胞匀浆用完整核协议尚未发表，并且在最发表评价 20 mL 的细胞悬液的被使用。为了适应这个经典的技术，以适应目前的工作与小规模的样品要求，我们提出氮汽蚀专为培养细胞的修改的协议。后氮气蚀、 匀浆分为可溶性 (S) 和膜 (P) 分数用差速离心法，首先低速旋转要移除细胞核和不间断的单元格，然后用一个高速旋转 (&#62; 100,000 x g) 来分隔可溶部分的膜。我们分析与 immunoblots 分离效率和比较氮汽蚀与其他机械破碎技术。我们也在氮空期间调查同质化缓冲区的渗透影响。

<table border="0" cellpadding="0" cellspacing="0" width="1015">
	<tbody>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Cell Disruption Vessel (45 mL)</td>
			<td style="width:181px;">Parr Instrument</td>
			<td style="width:145px;">4639</td>
			<td style="width:296px;">Nitrogen cavitation Bomb</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Dounce homogenizer (2 mL)</td>
			<td style="width:181px;">Kontes</td>
			<td style="width:145px;">885300-0002</td>
			<td style="width:296px;">Dounce pestle and tube</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">U-100 Insulin Syringe 28G&#189;</td>
			<td style="width:181px;">Becton Dickinson</td>
			<td style="width:145px;">329461</td>
			<td style="width:296px;">Needle</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Atg12 antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">271688</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">&#946;-actin antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">47778</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">&#946;-tubulin antibody</td>
			<td style="width:181px;">DSHB</td>
			<td style="width:145px;">E7-s</td>
			<td style="width:296px;">Mouse antibody, use at 1:5000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Calnexin antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">23954</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Calregulin antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">373863</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Catalase antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">271803</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">CIMPR antibody</td>
			<td style="width:181px;">Abcam</td>
			<td style="width:145px;">124767</td>
			<td style="width:296px;">Rabbit antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">EEA1 antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">137130</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">EGFR antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">373746</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">F0-ATPase antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">514419</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">F1-ATPase antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">55597</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Fibrillarin antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">374022</td>
			<td style="width:296px;">Mouse antibody, use at 1:200 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Golgin 97 antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">59820</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">HDAC1 antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">81598</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Hexokinase 1 antibody</td>
			<td style="width:181px;">Cell Signaling Technology</td>
			<td style="width:145px;">2024S</td>
			<td style="width:296px;">Rabbit antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Lamin A/C antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">376248</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">LAMP1 antibody</td>
			<td style="width:181px;">DSHB</td>
			<td style="width:145px;">H4A3-c</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Na+/K+ ATPase antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">48345</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Rab7 antibody</td>
			<td style="width:181px;">Abcam</td>
			<td style="width:145px;">137029</td>
			<td style="width:296px;">Rabbit antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Rab9 antibody</td>
			<td style="width:181px;">Thermo</td>
			<td style="width:145px;">MA3-067</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">RCAS1 antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">398052</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">RhoGDI antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">360</td>
			<td style="width:296px;">Rabbit antibody, use at 1:3000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Ribosomal protein S6 antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">74459</td>
			<td style="width:296px;">Mouse antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Sec61a antibody</td>
			<td style="width:181px;">Santa Cruz</td>
			<td style="width:145px;">12322</td>
			<td style="width:296px;">Goat antibody, use at 1:1000 dilution</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Thickwall Polycarbonate ultracentrifuge tube</td>
			<td style="width:181px;">Beckman Coulter</td>
			<td style="width:145px;">349622</td>
			<td style="width:296px;">Sample tube for ultracentrifugation</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">TLK-100.3 rotor</td>
			<td style="width:181px;">Beckman Coulter</td>
			<td style="width:145px;">349481</td>
			<td style="width:296px;">rotor for ultracentrifugation</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">Optima MAX High-Capacity Personal Ultracentrifuge</td>
			<td style="width:181px;">Beckman Coulter</td>
			<td style="width:145px;">364300</td>
			<td style="width:296px;">ultracentrifuge</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:393px;">cOmplete protease inhibitor cocktail tablets</td>
			<td>Roche</td>
			<td>11697498001</td>
			<td>protease inhibitors</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Cell Scrapers with 25cm Handle and 3.0cm Blade</td>
			<td>Corning</td>
			<td>353089</td>
			<td>large cell scraper</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Magnetic Stir Bar</td>
			<td>Fisher Scientific</td>
			<td>14-513-57SIX</td>
			<td>micro stir bar</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Ceramic-Top Magnetic Stirrer</td>
			<td>Fisher Scientific</td>
			<td>S504501AS</td>
			<td>magnetic stirrer</td>
		</tr>
	</tbody>
</table>

nitrogen cavitation and differential centrifugation allows for monitoring the distribution of peripheral membrane proteins in cultured cells

体外培养的细胞可用于研究蛋白质，包括周边的膜蛋白的亚细胞分布。基因编码荧光标记的蛋白质已经彻底改变了蛋白质的亚细胞分布的研究。然而，它很难量化与荧光显微镜、 分布，尤其是在部分胞浆蛋白时。此外，它往往是重要研究内源性蛋白。生化检测如 immunoblots 保持量化后亚细胞分离蛋白分布的金标准。虽然有旨在隔离胞浆或某些膜组分的商业工具，大部分的这些工具包基于使用洗涤剂，可能不适合学习轻松地提取膜的外周膜蛋白的提取。在这里我们目前细胞同质化洗涤剂无协议氮气蚀和随后的分离，胞浆和膜结合蛋白的超速离心。我们确认分离中可溶性细胞内细胞器和颗粒组分跨不同的细胞类型，并比较蛋白质提取中几种常见的非基于洗涤剂的机械同质化方法。中氮的一些优点空化是细胞中断到微妙的细胞器受到最小的物理和化学伤害的出色效率。氮空与超速离心法结合起来，是一个绝妙的方法来检查外周血膜蛋白之间胞浆的转变和膜组分。

图 2显示了分区从 PNS 细胞蛋白质水解成可溶性胞浆 (S) 或膜颗粒分数 (P)。我们研究了三种代表细胞从不同的细胞类型： 人胚肾 293 （上皮）、 NIH 3T3 （碱性成纤维细胞） 和白血病 （淋巴细胞）。Rho 鸟嘌呤分解抑制剂 （文章） 和阳离子独立甘露糖-6-磷酸受体 (CIMPR) 作为阳性对照为胞浆膜组分，分别。我们确认后氮空泡，胞浆和膜的高效分离 ~ 350 psi...

Watch this Scientific Journal Video about Nitrogen Cavitation and Differential Centrifugation Allows for Monitoring the Distribution of Peripheral Membrane Proteins in Cultured Cells at JoVE.com

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

在这里我们目前用于洗涤剂免费培养的哺乳动物细胞基于氮空和随后的分离，胞浆和膜结合蛋白的超速离心同质化的协议。此方法是理想的监测分区之间可溶性的外周膜蛋白和膜组分。

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 ...

Research

JoVE Journal

Biochemistry

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

科研

生物化学

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 ...

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

生物素化细胞表面蛋白的纯化<emRigicephalus microplus</em&gt;上皮细胞

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of ...

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 ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

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

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

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

实时测量核苷酸与完整功能性膜蛋白的结合

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

成熟水稻的横向切片（米 苜蓿L.）使用移液器吸头作为固定支架的扫描电子显微镜成像的内核

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Cell-Free Production of Proteoliposomes for Functional Analysis and Antibody Development Targeting Membrane Proteins

无细胞生产蛋白脂质体，用于功能分析和靶向膜蛋白的抗体开发

Membrane proteins play essential roles in a variety of cellular processes and perform vital functions. Membrane proteins are medically important in drug ...

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay (DRaCALA)

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay DRaCALA

用利甘德分析（DRACALA）的微分径向毛细管作用识别小配体的结合蛋白

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins ...