Name: nitrogen cavitation and differential centrifugation allows for monitoring the distribution of peripheral membrane proteins in cultured cells
Uploaded: 2017-08-18T14:00:00.000+00:00
Duration: 8 min 24 s
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 （以下简称低渗同质化缓冲区）。大多数的缓冲区可以改装为氮空 （见 讨论）。</li>
	<li>准备并冷藏 6 毫升的 x 每个样品在 4 ° C.添加蛋白酶抑制剂片新鲜之前使用 Phosphate-Buffered 盐渍 (PBS) 缓冲区 1。 
	注: 在本协议中使用的 PBS 缓冲包含 10 毫米 Na 2 HPO 4 在 pH 7.4，1.8 毫米 KH 2 PO 4，137 毫米氯化钠和氯化钾 2.7 毫米。</li>
	<li>准备并冷藏 4 毫升的每个样品在 4 ° C.添加一个蛋白酶抑制剂平板电脑只是之前使用的增溶性缓冲。 
	注: 本协议中使用的增溶作用缓冲区是 1 x Radioimmunoprecipitation 测定 （马国） 缓冲区，该缓冲区由 25 毫米三在 pH 7.4，150 毫米氯化钠、 0.1 %sds、 脱氧胆酸钠 0.5%，1 %np-40。请参阅 4.6 注释。</li>
</ol> 2。细胞收获 <ol><li>成长 2 x 10 7-10 x 10 9 组织培养细胞与推荐文化传媒为单元格类型。通常情况下，一个 15 厘米菜产量 2 x 10 7 90%融合 ( 图 1) 在 DMEM 培养的人胚肾 293 细胞。</li>
	<li>删除由真空生长培养基。</li>
	<li>对于贴壁细胞，将培养皿放在冰上、 洗直接上培养皿轻轻地用冷冻同质化缓冲两次 （每次洗涤 15 cm 培养皿每 10 毫升缓冲区） 的单元格和用在适当的卷的大细胞刮刀收集细胞同质化缓冲区;对于悬浮细胞，收集和洗细胞颗粒冷冻同质化缓冲区中两次 （每 50 毫升文化每次洗涤 10 毫升缓冲） 500 x g 自旋在 4 ° C 5 分钟，并洗的细胞颗粒在适当体积的冰上的同质化缓冲区。 注意: 卷应基于预期的试验以及细胞破坏炸弹所允许的最小/最大卷蛋白质浓度的要求。一般准则是: 2-10 x 10 7 细胞/毫升，或约 10 卷单元格的颗粒。本议定书为三个 15 厘米菜人胚肾 293 细胞的 2 毫升的同质化缓冲区优化。</li>
</ol> 3。氮空 <ol><li>清洁及冷冻炸弹在冰浴上轰动转移细胞悬浮液板。 
	警告: 炸弹有高压、 低温、 氮气 &#8211; 穿适当的个人防护 .</li>
	<li>微磁搅拌棒放置炸弹，并开启搅拌盘保持悬浮均匀性。</li>
	<li>添加一个蛋白酶抑制剂平板电脑到暂停并关闭按制造商炸弹 &#39; s 指令。</li>
	<li>逐渐加压与氮气罐按制造商炸弹 &#39; s 指令直到炸弹压力表读取 300 到 600 磅/平方英寸。关闭所有阀门和断开氮罐。 
	注: 所需的压力可能随细胞类型。在这里我们进行 350 到 400 磅/平方英寸的气蚀人胚肾 293、 NIH 3T3 和白血病细胞。</li>
	<li>等待 20 分钟，使氮溶解并达到平衡细胞内的。</li>
	<li>删除多余的水排出阀使用毛巾布周围。打开排出阀轻轻地实现匀浆滴状释放和收集在预冷 15 毫升管。 
	注: 在集合的末尾附近会有井喷式的匀浆，气体将乘着嘶嘶的声音。请确保气体不原因以前收集气蚀余量射管 （因此使用 15 毫升管而不是 1.5 毫升管）。一旦开始冲刺，关闭排出阀，打开氮气入口阀突然减压炸弹并实现空化在炸弹中剩余的单元格。开放为炸弹气蚀余量恢复和大扫除。 
	注意: 最后空化应顶部有乳白色泡沫。轻轻地用针提示允许泡沫消退之前离心搅拌。 
	注意: 通过相差显微镜检查，以确定同质化效率检查空化。加一 15 微升滴流于表面的显微镜幻灯片，盖上盖玻片。重复步骤 3.4-3.6 只当太多的不间断的细胞检测到含 20 X 目的。 
	注意: 如果同质化缓冲区不包含 EDTA 或 EGTA，将它添加到收集在出院后终浓度为 1 毫米，5 分钟内气蚀余量。</li>
</ol> 4。Cytosolic 和膜组分的分离 <ol><li>离心机在 500 x 空化 g 10 分钟在 4 ° C，若要删除不间断的细胞和细胞核。 
	注: 重复离心步骤，直到没有可见的颗粒产生和收集后核清 (PNS)，而避免泡沫浮在上面。如有必要再离心泡沫进一步收集和合并 PNS，( 图 1)。</li>
	<li>处理 PNS 如愿获得分数的兴趣。为了分离胞浆和膜组分，转移到离心管中三七总皂苷和执行所需的超速离心法。为优化本议定书 &#60; 3.5 毫升样品在聚碳酸酯离心管和超速离心法在 350,000 x g 为 1 h 在 4 ° C.</li>
	<li>收集上清液 （S 分数） 使用 1 毫升吸管。</li>
	<li>仔细冲洗用冷的 PBS 3 毫升颗粒没有打扰它。删除由真空 PBS。 
	注意: 如果胞浆蛋白膜组分的污染是比样品损失更大的关注，在 3 毫升的冷 PBS 和 re 离心步骤 4.2 悬小球。删除由真空 PBS。</li>
	<li>悬小球完全在含洗涤剂的增溶作用缓冲区的选择适当的卷。为了实现单元格等价，用作同样体积的增溶作用缓冲区胞浆。 
	注意: 我们建议使用 1 x RIPA 缓冲区作为增溶缓冲区高效膜蛋白萃取。如果没有下游的测定需要膜只是小部分，最大的膜蛋白提取使用 1 x laemmli 样品缓冲液。 
	注意: 我们建议取出并转移到清洁管管转子在 4 ° C，最大的膜蛋白提取的增溶缓冲区中的颗粒。 
	注意: 如果颗粒太黏太多脂） 有效地将从 the 离心管增溶缓冲区中的，我们建议管理单元在液氮中冷冻颗粒和快速取出离心管中的颗粒用迷你金属锅铲前颗粒融化。 
	注: 另外，膜颗粒可以只是再悬浮和不溶解在非含洗涤剂的缓冲区来产生几分 P 膜囊泡悬浮体系，在这情况下离心步骤在 4.6 不是必需。这种 P 分数是有用的当调查功能，如依赖膜协会的酶活动。</li>
	<li>将充分溶解的颗粒悬浮液从步 4.5 20,000 x g 在 10 分钟的台式离心机离心在 4 ° C.收集上清液使用 （P 分数） 1ml 移液器和弃沉淀 （不溶性血脂）。</li>
	<li>执行所需的化验，如印迹与胞浆和 （或） 膜的分数，或将其保存在-80 ° C 供将来使用。</li>
</ol>

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作者宣称，他们有没有经济利益的竞争。

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><tbody><tr><td>Cell Disruption Vessel (45 mL)</td><td>Parr Instrument</td><td>4639</td><td>Nitrogen cavitation Bomb</td></tr><tr><td>Dounce homogenizer (2 mL)</td><td>Kontes</td><td>885300-0002</td><td>Dounce pestle and tube</td></tr><tr><td>U-100 Insulin Syringe 28G&amp;frac12;</td><td>Becton Dickinson</td><td>329461</td><td>Needle</td></tr><tr><td>Atg12 antibody</td><td>Santa Cruz</td><td>271688</td><td>Mouse antibody, use at 1:1000 dilution</td></tr><tr><td>&amp;beta;-actin antibody</td><td>Santa Cruz</td><td>47778</td><td>Mouse antibody, use at 1:1000 dilution</td></tr><tr><td>&amp;beta;-tubulin antibody</td><td>DSHB</td><td>E7-s</td><td>Mouse antibody, use at 1:5000 dilution</td></tr><tr><td>Calnexin antibody</td><td>Santa Cruz</td><td>23954</td><td>Mouse antibody, use at 1:1000 dilution</td></tr><tr><td>Calregulin antibody</td><td>Santa Cruz</td><td>373863</td><td>Mouse antibody, use at 1:1000 dilution</td></tr><tr><td>Catalase antibody</td><td>Santa Cruz</td><td>271803</td><td>Mouse antibody, use at 1:1000 dilution</td></tr><tr><td>CIMPR antibody</td><td>Abcam</td><td>124767</td><td>Rabbit antibody, use at 1:1000 dilution</td></tr><tr><td>EEA1 antibody</td><td>Santa Cruz</td><td>137130</td><td>Mouse antibody, use at 1:1000 dilution</td></tr><tr><td>EGFR antibody</td><td>Santa Cruz</td><td>373746</td><td>Mouse antibody, use at 1:1000 dilution</td></tr><tr><td>F&lt;sub&gt;0&lt;/sub&gt;-ATPase antibody</td><td>Santa Cruz</td><td>514419</td><td>Mouse antibody, use at 1:1000 dilution</td></tr><tr><td>F&lt;sub&gt;1&lt;/sub&gt;-ATPase antibody</td><td>Santa Cruz</td><td>55597</td><td>Mouse antibody, use at 1:1000 dilution</td></tr><tr><td>Fibrillarin antibody</td><td>Santa Cruz</td><td>374022</td><td>Mouse antibody, use at 1:200 dilution</td></tr><tr><td>Golgin 97 antibody</td><td>Santa Cruz</td><td>59820</td><td>Mouse antibody, use at 1:1000 dilution</td></tr><tr><td>HDAC1 antibody</td><td>Santa Cruz</td><td>81598</td><td>Mouse antibody, use at 1:1000 dilution</td></tr><tr><td>Hexokinase 1 antibody</td><td>Cell Signaling Technology</td><td>2024S</td><td>Rabbit antibody, use at 1:1000 dilution</td></tr><tr><td>Lamin A/C antibody</td><td>Santa Cruz</td><td>376248</td><td>Mouse antibody, use at 1:1000 dilution</td></tr><tr><td>LAMP1 antibody</td><td>DSHB</td><td>H4A3-c</td><td>Mouse antibody, use at 1:1000 dilution</td></tr><tr><td>Na&lt;sup&gt;+&lt;/sup&gt;/K&lt;sup&gt;+&lt;/sup&gt; ATPase antibody</td><td>Santa Cruz</td><td>48345</td><td>Mouse antibody, use at 1:1000 dilution</td></tr><tr><td>Rab7 antibody</td><td>Abcam</td><td>137029</td><td>Rabbit antibody, use at 1:1000 dilution</td></tr><tr><td>Rab9 antibody</td><td>Thermo</td><td>MA3-067</td><td>Mouse antibody, use at 1:1000 dilution</td></tr><tr><td>RCAS1 antibody</td><td>Santa Cruz</td><td>398052</td><td>Mouse antibody, use at 1:1000 dilution</td></tr><tr><td>RhoGDI antibody</td><td>Santa Cruz</td><td>360</td><td>Rabbit antibody, use at 1:3000 dilution</td></tr><tr><td>Ribosomal protein S6 antibody</td><td>Santa Cruz</td><td>74459</td><td>Mouse antibody, use at 1:1000 dilution</td></tr><tr><td>Sec61a antibody</td><td>Santa Cruz</td><td>12322</td><td>Goat antibody, use at 1:1000 dilution</td></tr><tr><td>Thickwall Polycarbonate ultracentrifuge tube</td><td>Beckman Coulter</td><td>349622</td><td>Sample tube for ultracentrifugation</td></tr><tr><td>TLK-100.3 rotor</td><td>Beckman Coulter</td><td>349481</td><td>rotor for ultracentrifugation</td></tr><tr><td>Optima MAX High-Capacity Personal Ultracentrifuge</td><td>Beckman Coulter</td><td>364300</td><td>ultracentrifuge</td></tr><tr><td>cOmplete protease inhibitor cocktail tablets</td><td>Roche</td><td>11697498001</td><td>protease inhibitors</td></tr><tr><td>Cell Scrapers with 25cm Handle and 3.0cm Blade</td><td>Corning</td><td>353089</td><td>large cell scraper</td></tr><tr><td>Magnetic Stir Bar</td><td>Fisher Scientific</td><td>14-513-57SIX</td><td>micro stir bar</td></tr><tr><td>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 ...

nitrogen-cavitation-differential-centrifugation-allows-for-monitoring

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

16.0K 次观看.  New York University. 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。

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

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

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. This method utilizes hypotonic buffers, digitonin, mechanical lysis and differential centrifugation to isolate the cytoplasm, mitochondria and plasma membrane. The process can be scaled to accommodate the needs of researchers, is inexpensive and straightforward. This method will allow researchers to determine protein localization in cells without specialized centrifuges and without the use of commercial kits, both of which can be prohibitively expensive. We have successfully used this method to separate cytosolic, plasma membrane and mitochondrial proteins in the human monocyte cell line U937.

Here, we present a protocol to isolate the plasma membrane, cytoplasm and mitochondria of U937 cells without the use of high-speed centrifugation. This technique can be used to purify subcellular fractions for subsequent examination of protein localization via immunoblotting.

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. ...

科研

生物化学

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal transduction; from sensing environmental signals to biological response; from metabolic regulation to developmental control. Accurate structural information of protein-protein interactions is crucial for understanding the molecular mechanisms of membrane protein complexes and for the design of highly specific molecules to modulate these proteins. Many in vivo and in vitro approaches have been developed for the detection and analysis of protein-protein interactions. Among them the structural biology approach is unique in that it can provide direct structural information of protein-protein interactions at the atomic level. However, current membrane protein structural biology is still largely limited to detergent-based methods. The major drawback of detergent-based methods is that they often dissociate or denature membrane protein complexes once their native lipid bilayer environment is removed by detergent molecules. We have been developing a&#160;native cell membrane nanoparticle system for membrane protein structural biology. Here, we demonstrate the use of this system in the analysis of protein-protein interactions on the cell membrane with a case study of the oligomeric state of AcrB.

Presented here is a protocol for the determination of oligomeric state of membrane proteins that utilizes a native cell membrane nanoparticle system in conjunction with electron microscopy.

用于膜蛋白-蛋白质相互作用分析的原生细胞膜纳米粒子系统

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and isopycnic density gradient centrifugation. The major advantage of this procedure is that it does not rely on the sole use of solubilizing detergents to obtain subcellular fractions. This makes it possible to separate the plasma membrane from other membrane-bound organelles of the cell. This procedure will facilitate the determination of protein localization in cells with a reproducible, scalable, and selective method. This method has been successfully used to isolate cytosolic, nuclear, mitochondrial, and plasma membrane proteins from the human monocyte cell line, U937. Although optimized for this cell line, this procedure may serve as a suitable starting point for the subcellular fractionation of other cell lines. Potential pitfalls of the procedure and how to avoid them are discussed as are alterations that may need to be considered for other cell lines.

This fractionation protocol will allow researchers to isolate cytoplasmic, nuclear, mitochondrial, and membrane proteins from mammalian cells. The latter two subcellular fractions are further purified via isopycnic density gradient.

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 ...

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)&#8211;Cell Interaction and the Resultant Bioeffects at the Single-cell Level

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass substrate, that precisely controls the generation of tandem bubbles and individual cells patterned nearby with well-defined locations and shapes. We then demonstrate the generation of tandem bubbles by using two pulsed lasers illuminating a pair of gold dots with a few-microsecond time delay. We visualize the bubble-bubble interaction and jet formation by high-speed imaging and characterize the resultant flow field using particle image velocimetry (PIV). Finally, we present some applications of this technique for single cell analysis, including cell membrane poration with macromolecule uptake, localized membrane deformation determined by the displacements of attached integrin-binding beads, and intracellular calcium response from ratiometric imaging. Our results show that a fast and directional jetting flow is produced by the tandem bubble interaction, which can impose a highly localized shear stress on the surface of a cell grown in close proximity. Furthermore, different bioeffects can be induced by altering the strength of the jetting flow by adjusting the standoff distance from the cell to the tandem bubbles.

A microfluidic chip was fabricated to produce pairs of gold dots for tandem bubble generation and fibronectin-coated islands for single-cell patterning nearby. The resultant flow field was characterized by particle image velocimetry and was employed to study various bioeffects, including cell membrane poration, membrane deformation, and intracellular calcium response.

用的表面图案化微流体系统用于在单细胞水平调查空泡（S） - 细胞的相互作用及产生的生物效应

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass ...

生物工程

Rapid Assessment of Membrane Protein Quality by Fluorescent Size Exclusion Chromatography

During membrane protein structural elucidation and biophysical characterization, it is common to trial numerous protein constructs containing different tags, truncations, deletions, fusion partner insertions, and stabilizing mutations to find one that is not aggregated after extraction from the membrane. Furthermore, buffer screening to determine the detergent, additive, ligand, or polymer that provides the most stabilizing condition for the membrane protein is an important practice. The early characterization of membrane protein quality by fluorescent size exclusion chromatography provides a powerful tool to assess and rank different constructs or conditions without the requirement for protein purification, and this tool also minimizes the sample requirement. The membrane proteins must be fluorescently tagged, commonly by expressing them with a GFP tag or similar. The protein can be solubilized directly from whole cells and then crudely clarified by centrifugation; subsequently, the protein is passed down a size exclusion column, and a fluorescent trace is collected. Here, a method for running FSEC and representative FSEC data on the GPCR targets sphingosine-1-phosphate receptor (S1PR1) and serotonin receptor (5HT2AR) are presented.

The present protocol describes a procedure to perform fluorescent size exclusion chromatography (FSEC) on membrane proteins to assess their quality for downstream functional and structural analysis. Representative FSEC results collected for several G-protein coupled receptors (GPCRs) under detergent-solubilized and detergent-free conditions are presented.

通过荧光体积排阻色谱快速评估膜蛋白质量

During membrane protein structural elucidation and biophysical characterization, it is common to trial numerous protein constructs containing different ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

采用组合运用各种方法，定义质膜交付在轴突分支和神经元形态发生中的作用

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

神经科学

Neutron Spin Echo Spectroscopy as a Unique Probe for Lipid Membrane Dynamics and Membrane-Protein Interactions

Lipid bilayers form the main matrix of cell membranes and are the primary platform for nutrient exchange, protein-membrane&#160;interactions, and viral budding, among other vital cellular processes. For efficient biological activity, cell membranes should be rigid enough to maintain the integrity of the cell and its compartments yet fluid enough to allow membrane components, such as proteins and functional domains, to diffuse and interact. This delicate balance of elastic and fluid membrane properties, and their impact on biological function, necessitate a better understanding of collective membrane dynamics over mesoscopic length and time scales of key biological processes, e.g., membrane deformations and protein binding events. Among the techniques that can effectively probe this dynamic range is neutron spin echo (NSE) spectroscopy. Combined with deuterium labeling, NSE can be used to directly access bending and thickness fluctuations as well as mesoscopic dynamics of select membrane features. This paper provides a brief description of the NSE technique and outlines the procedures for performing NSE experiments on liposomal membranes, including details of sample preparation and deuteration schemes, along with instructions for data collection and reduction. The paper also introduces data analysis methods used to extract key membrane parameters, such as the bending rigidity modulus, area compressibility modulus, and in-plane viscosity. To illustrate the biological importance of NSE studies, select examples of membrane phenomena probed by NSE are discussed, namely, the effect of additives on membrane bending rigidity, the impact of domain formation on membrane fluctuations, and the dynamic signature of membrane-protein interactions.

This paper describes the protocols for sample preparation, data reduction, and data analysis in neutron spin echo (NSE) studies of lipid membranes. Judicious deuterium labeling of lipids enables access to different membrane dynamics on mesoscopic length and time scales, over which vital biological processes occur.

中子自旋回声光谱学作为脂膜动力学和膜-蛋白相互作用的独特探针

Lipid bilayers form the main matrix of cell membranes and are the primary platform for nutrient exchange, protein-membrane&#160;interactions, and viral ...

生物学

用于微生物群多组学的增强细菌细胞外囊泡分离

Isolation and Purification of Bacterial Extracellular Vesicles from Human Feces Using Density Gradient Centrifugation

Bacterial extracellular vesicles (BEVs) are nanovesicles derived from bacteria that play an active role in bacteria-bacteria and bacteria-host communication, transferring bioactive molecules such as proteins, lipids, and nucleic acids inherited from the parent bacteria. BEVs derived from the gut microbiota have effects within the gastrointestinal tract and can reach distant organs, resulting in significant implications for physiology and pathology. Theoretical investigations that explore the types, quantities, and roles of BEVs derived from human feces are crucial for understanding the secretion and function of BEVs from the gut microbiota. These investigations also necessitate an improvement in the current strategy for isolating and purifying BEVs.
This study optimized the isolation and purification process of BEVs by establishing two density gradient centrifugation (DGC) modes: Top-down and Bottom-up. The enriched distribution of BEVs was determined in fractions 6 to 8 (F6-F8). The effectiveness of the approach was evaluated based on particle morphology, size, concentration, and protein content. The particle and protein recovery rates were calculated, and the presence of specific markers was analyzed to compare the recovery and purity of the two DGC modes. The results indicated that the Top-down centrifugation mode had lower contamination levels and achieved a recovery rate and purity similar to that of the Bottom-up mode. A centrifugation time of 7 h was sufficient to achieve a fecal BEV concentration of 108/mg.
Apart from feces, this method could be applied to other body fluid types with proper modification according to the differences in components and viscosity. In conclusion, this detailed and reliable protocol would facilitate the standardized isolation and purification of BEVs and thus, lay a foundation for subsequent multi-omics analysis and functional experiments.

作者聚焦：加速细菌细胞外囊泡分离和异质性的研究 

This study describes a method to isolate and purify bacterial extracellular vesicles (BEVs) enriched from human feces via density gradient centrifugation (DGC), identifies the physical characteristics of BEVs from morphology, particle size, and concentration, and discusses the potential applications of the DGC approach in clinical and scientific research.

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

摘要

探索更多视频

此视频中的章节

高速离心条件下 u937 细胞的细胞分裂

用于膜蛋白-蛋白质相互作用分析的原生细胞膜纳米粒子系统

通过等密度梯度纯化对U937细胞进行细胞分级分离

用的表面图案化微流体系统用于在单细胞水平调查空泡（S） - 细胞的相互作用及产生的生物效应

通过荧光体积排阻色谱快速评估膜蛋白质量

采用组合运用各种方法，定义质膜交付在轴突分支和神经元形态发生中的作用

中子自旋回声光谱学作为脂膜动力学和膜-蛋白相互作用的独特探针

使用密度梯度离心从人粪便中分离和纯化细菌胞外囊泡

使用密度梯度离心从人粪便中分离和纯化细菌胞外囊泡

使用密度梯度离心从人粪便中分离和纯化细菌胞外囊泡