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>

<ol>
	<li>Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteins+on+the+move:+insights+gained+from+fluorescent+protein+technologies.">Proteins on the move: insights gained from fluorescent protein technologies.</a> Nat Rev Mol Cell Biol. 12 (10), 656-668 (2011).</li><li>Bunger, S., Roblick, U. J., Habermann, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+five+commercial+extraction+kits+for+subsequent+membrane+protein+profiling.">Comparison of five commercial extraction kits for subsequent membrane protein profiling.</a> Cytotechnology. 61 (3), 153-159 (2009).</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=Disruption+of+cultured+cells+by+nitrogen+cavitation.">Disruption of cultured cells by nitrogen cavitation.</a> Cold Spring Harb Protoc. (11), (2010).</li><li>Klempner, M. S., Mikkelsen, R. B., Corfman, D. H., Andre-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+plasma+membranes.+I.+High-yield+purification+of+human+neutrophil+plasma+membrane+vesicles+by+nitrogen+cavitation+and+differential+centrifugation.">Neutrophil plasma membranes. I. High-yield purification of human neutrophil plasma membrane vesicles by nitrogen cavitation and differential centrifugation.</a> J Cell Biol. 86 (1), 21-28 (1980).</li><li>Philips, M. R., 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=Low+molecular+weight+GTP-binding+proteins+in+human+neutrophil+granule+membranes.">Low molecular weight GTP-binding proteins in human neutrophil granule membranes.</a> Journal of Biological Chemistry. 266, 1289-1298 (1991).</li><li>Wallach, D. F., Soderberg, J., Bricker, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phospholipides+of+Ehrlich+ascites+carcinoma+cells:+composition+and+intracellular+distribution.">The phospholipides of Ehrlich ascites carcinoma cells: composition and intracellular distribution.</a> Cancer Res. 20, 397-402 (1960).</li><li>Hunter, M. J., Commerford, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure+homogenization+of+mammalian+tissues.">Pressure homogenization of mammalian tissues.</a> Biochim Biophys Acta. 47, 580-586 (1961).</li><li>Wallach, D. F., Kamat, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+and+Cytoplasmic+Membrane+Fragments+from+Ehrlich+Ascites+Carcinoma.">Plasma and Cytoplasmic Membrane Fragments from Ehrlich Ascites Carcinoma.</a> Proc Natl Acad Sci U S A. 52, 721-728 (1964).</li><li>Birckbichler, P. 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. P., 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=Rap1-interacting+adapter+molecule+(RIAM)+associates+with+the+plasma+membrane+via+a+proximity+detector.">Rap1-interacting adapter molecule (RIAM) associates with the plasma membrane via a proximity detector.</a> J Cell Biol. , (2012).</li><li>Zhou, M., 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=VPS35+binds+farnesylated+N-Ras+in+the+cytosol+to+regulate+N-Ras+trafficking.">VPS35 binds farnesylated N-Ras in the cytosol to regulate N-Ras trafficking.</a> J Cell Biol. 214 (4), 445-458 (2016).</li><li>Sim, D. S., Dilks, J. R., Flaumenhaft, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelets+possess+and+require+an+active+protein+palmitoylation+pathway+for+agonist-mediated+activation+and+in+vivo+thrombus+formation.">Platelets possess and require an active protein palmitoylation pathway for agonist-mediated activation and in vivo thrombus formation.</a> Arterioscler Thromb Vasc Biol. 27 (6), 1478-1485 (2007).</li><li>Pace, P. E., Peskin, A. V., Han, M. H., Hampton, M. B., Winterbourn, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperoxidized+peroxiredoxin+2+interacts+with+the+protein+disulfide-+isomerase+ERp46.">Hyperoxidized peroxiredoxin 2 interacts with the protein disulfide- isomerase ERp46.</a> Biochem J. 453 (3), 475-485 (2013).</li><li>Annis, M. G., 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=Endoplasmic+reticulum+localized+Bcl-2+prevents+apoptosis+when+redistribution+of+cytochrome+c+is+a+late+event.">Endoplasmic reticulum localized Bcl-2 prevents apoptosis when redistribution of cytochrome c is a late event.</a> Oncogene. 20 (16), 1939-1952 (2001).</li><li>Berkowitz, S. A., Wolff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+calcium+sensitivity+of+tubulin+polymerization.+The+contributions+of+temperature,+tubulin+concentration,+and+associated+proteins.">Intrinsic calcium sensitivity of tubulin polymerization. The contributions of temperature, tubulin concentration, and associated proteins.</a> J Biol Chem. 256 (21), 11216-11223 (1981).</li></ol>

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。氮空还提供更一致的结果，在整个示例统一应用同样的破坏性力量可以转载相同的压力。而且在此基础上，每个单元格只经历了中断过程为一次，亚细胞成分，因此，不经常被暴露变量破坏性的力量。这就限制了细胞器出土文物碎片。氧化的活性细胞成分引起中断的关注也减轻，如氮用于饱和的细胞悬液，无补充氧气。受限制的物理和化学应力征收氮空使它理想的技术，研究活性酶和脆弱的细胞器和可重复性的这种同质化技术非常适合量化。
氮空还提供了很大的灵活性，在样本大小、 组成的同质化缓冲区和细胞器完整性的程度。为了加强同质化，只需要部署压力容器的更大的容量，而不会牺牲速度、 方便或减压的效率。任何同质化缓冲区可以用于氮汽蚀;缓冲区的选择可以为符合每次实验的要求量身定做。一个可以设计匹配 （例如高钾、 低钠） 尽量减少改建细胞内液中细胞器的同质化缓冲区。例如，我们开发了"放松"的缓冲区的最小化体外肌动蛋白聚合，从而协助从粒细胞5收集完整的胞浆囊泡。也可以修改缓冲区的渗透和离子特征，以控制细胞同质化程度。此外，可以通过调节氮压力控制侵略性细胞的破坏。适度的压力减少了维护细胞核、 线粒体和其他细胞器完好状态，而高压扰乱这些细胞器的破坏性力量。布洛克et al。报告成功干扰大鼠嗜碱粒细胞白血病细胞的同时保持大多数原子核完整在 350 psi 与低渗缓冲10，形成氮气压力在我们当前的协议中使用的基础。
氮空蚀的限制包括事实同质化是统一整个样本和不同的膜结构可能产生囊泡的大小差不多;这可以通过率纬向密度梯度离心法分离复杂细胞膜、 细胞质、 光滑的微粒体、 高尔基膜进一步的分离。另一个担忧是细胞器同质化从内部破裂后变得相对脆弱。因此，这种方法需要精心的优化，以防止在后续操作中的细胞器破损。我们的目的是要解决这些问题在本研究中的一些。
分离的胞浆膜组分用离心法探索了在有限的报告，详细说明分区蛋白质组分与少量的蛋白质标记15，16。我们的方法生成可溶性和膜结合蛋白，粗分离，并且不涉及特定细胞器的分离。然而，它仍然是有效的关切，均匀不同膜产生的空化泡的超速离心法，可能会影响分离效率。我们提出一项全面调查隔离的 immunoblots 和分区的常见细胞内细胞器后分馏与我们的协议。不出所料，完全是像文章纯粹胞浆蛋白中的可溶部分和跨膜蛋白对细胞膜 (PM) 如 EGFR 和 Na+/K+ atp 酶，被发现只有于颗粒。对 ER、 线粒体、 高尔基体、 溶酶体等细胞器的跨膜蛋白也恢复在颗粒，确认从不同细胞膜和细胞器的内在膜蛋白可以与可溶性蛋白成功地分开我们的方法。这个基本的验证是外围的膜蛋白的进一步分析的关键是本议定书的主要目的。
最外围的膜蛋白存在于胞浆和膜组分。虽然这常常是其本地化的真实再现，它可能在某些情况下代表出土文物重新分配这些蛋白质从细胞质膜表面可溶部分在同质化的过程中，因为其协会与膜的强度是不一样强烈的内在膜蛋白。虽然氮空通过揭露细胞对一次性的、 温柔的中断最小化潜在的再分配，应当考虑这类文物的生化分离，并侧重于在分区之间胞浆蛋白改变的和实验条件跨膜组分。尽管如此外, 周膜蛋白在不同阶段的内涵成熟考试是用这种方法。EEA1，介导停靠的涂覆网格蛋白小泡到早期的内小体，Rab5 效应器定位于细胞质表面的早期内吞小体和失而复得主要在颗粒分数。晚体膜相关的 Rab7 和 Rab9 蛋白也在球团分数。然而，仍那里数量可观的 Rab7/9 在胞浆内。因为 RabGDI 是能够与异戊烯基 Rabs 交互和呈现这些否则为不溶性蛋白质胞浆，Rab 蛋白往往要比其他体膜相关外围膜蛋白更胞浆。
表征的核和细胞器的诚信与同质化氮空穴现象，我们 immunoblotted 的管腔及细胞质车厢分离的组分。在此分析中，calregulin 是完全释放到可溶，表明 ER 对窗体微粒体破坏、 从而泄漏管腔内容。鉴于那个一半在真核细胞的膜总面积包围 ER 的迷宫般的空间，也就不奇怪，囊状或管状结构的庞大网络未能在氮气泡沫扩张期间保持不变。另一方面，我们的线粒体完整后空化的分析显示明显的依赖性的细胞类型，如我们观察到 F1-atp 酶，是静脉与线粒体，胞浆的内在膜蛋白（人胚肾 293 和白血病 ~ 35%，2%为 NIH 3T3） 的程度。这表明，氮在 350 psi 与低渗缓冲区空并不能保证线粒体的完整。事实上，别人有报道，要维持线粒体，空化应执行在压力低于 150 psi17。相比之下，我们发现，过氧化氢酶仍然在肠腔内的过氧化物酶体。有趣的是，观察到了核蛋白质的三七总皂苷和颗粒的分数。具体来说，HDAC1，目前整个核质，组蛋白去乙酰化酶是缺席在可溶部分，指示核的完整性不被破坏。这表明，核膜不会破裂的气蚀，正如人们料想 HDAC1 释放到水溶性在这种情况下的乐分数。它仍然可能核未完全删除三七总皂苷制备过程和蛋白质超速离心法后，留在球团。这种可能性进一步支持了这一事实没有 DNA 渗漏进行检测在可溶部分。因此，我们得出结论，在加镁2 + ~ 350 磅/平方英寸的气蚀过程中使用低渗同质化缓冲区是能够保持核的完整性，在多个单元格的行。
我们的研究结果也表明核糖体收集完全在膜颗粒分数，暗示甚至游离核糖体细胞质中，绑定到 ER 膜可以完全沉淀后的蔗糖垫层缓冲区中在为 1 h.同样，识别 12 细胞自噬相关蛋白 (Atg12) 的 350,000 x g 离心-Atg5 共轭作为标记噬，揭示了颗粒组分对严格隔离。它在胞浆内的存在可能引起共价键的 Atg12 和 Atg5 之前噬形成的事实。细胞骨架蛋白很难估计，因为体外聚合。松弛缓冲，可减少这种现象却在这里描述的协议中，微管蛋白可能聚合体外后的钙 18 螯合作用。我们的筹备工作在暗示聚合，而肌动蛋白发现在 P 和 S 组分的颗粒在微管蛋白。
在同质化效率表征氮汽蚀的潜在优势，我们回顾了一些常用的机械破碎技术。不评价方法优化的组织样本，例如搅拌器或砂浆/杵。因为它就很难同时保持内部的细胞器完好破坏细胞膜表面，超声波也排除在外。我们的分析侧重于下列方法: 冷冻-解冻，针通道和 Dounce 同质化，因为它们很容易执行，不需要特殊的设备，如滚珠均质机。白血病细胞进行这种分析由于其体积小，这使它们难以有效地与传统液的剪切技术依赖间隙，如针通道和 Dounce 同质化。我们判断的恢复总蛋白浓度，发现氮空蚀和冻融等单元格大小独立中断方法的确表明蛋白质提取效率出众。类似的分析，较大尺寸的细胞发现差异较小，在同质化的效率，虽然氮空这仍然优于替代物理方法测试 （数据未显示）。
许多胞质，内涵和细胞骨架蛋白进行了有效地回收利用所有的测试方法。相比之下，对 ER 和高尔基体蛋白质最佳恢复使用氮气气蚀。略低产量的 F1-atp 酶活性氮汽蚀相对其他方法可能反映事实线粒体是更有可能在空化期间保持不变，因此更有效去除速度慢自旋在习惯于生成的期票。观察那己糖激酶 1，外围膜蛋白发现两者在胞浆内和与线粒体外膜，表现以相反的方式相对于 F1-atp 酶可能反映了协会的统一化己糖激酶 1 相对于 F1-atp 酶，线粒体内封存。重要的是，虽然能干扰细胞与可比效率，替代机械破碎方法给了相对较差的某些外围膜蛋白 （己糖激酶 1 和 RAS） 恢复。因此，空化是选择的我们同质化技术以作调查周边的膜蛋白的分区。因为 Dounce 同质化广泛用于准备完整的细胞核，我们用于 Dounce 作为基准评估核完整性各个其他机械破碎方法。在我们的分析，类似的回收 HDAC1 蛋白质水平确认核依然完整地保留在白血病组织匀浆法制备氮汽蚀与低渗缓冲 ~ 350 磅/平方英寸。
很明显，在氮空化过程中所使用的同质化缓冲区可以显著影响中断效率和细胞器完整性。虽然同质化缓冲区组成应优化目标应用程序的要求，它值得注意的研究人员表演的动物组织氮减压与低渗透压通过使用缓冲区来提取核内容。猎人和康墨显示细胞核肿胀和破裂时的解决办法是稀的和核保存使用等渗解决方案7 （这可以通过添加无机盐如氯化钠或有机溶质如实现时蔗糖或甘油）。用于隔离除了从哺乳动物细胞的细胞核的细胞内细胞器的标准等渗缓冲区是 0.25 M 蔗糖含有 1 毫米 EDTA 和 ph 值为 7.0-7.6 缓冲与三、 复或酰胺。但并不是所有文化细胞可以有效地匀都浆于一体的等渗的缓冲区。低渗缓冲区 (通常 10 毫米席复，等) 通常用于提供必要的渗透胁迫对某些单元格类型来实现完整的同质化。然而，如前所述，倾向于低渗缓冲区时 EDTA 是包括脆弱和易出现漏损的 DNA 呈现细胞核。若要促进核的完整性，我们替换 EDTA KCl 和低浓度的二价阳离子如氯化镁2或硫酸镁4。Mg2 +通常是首选 Ca2 +因为后者可以激活某些磷脂酶和蛋白酶和抑制 RNA 聚合酶。空化完成后，EDTA 或 EGTA 可以重新添加到组织匀浆，螯合阳离子，如果需要抑制金属蛋白酶。
白血病细胞显示细胞核细胞质比相对较大，使他们理想的单元格行研究核的完整性。比较到辅以 NaCl 的等渗缓冲区时，通过使用低渗缓冲区的同质化效率的提高是最小。然而，辅以蔗糖等渗缓冲区中的空化恢复大约一半利用低渗缓冲区对应提取的蛋白质。这是似乎不会直接造成的无量，和使用氯化钠和利用蔗糖实现无量之间的差距，需要进一步调查。一个可能的解释是盐破坏蛋白质间的静电相互作用，而蔗糖不。因此，即使在相同的强健性，NaCl 具有与蔗糖缺乏的蛋白质提取有关的活动。With 方面保持核的完整性，我们发现增加的 fibrillarin 蛋白用等渗的缓冲区，尽管等渗缓冲区理论上都更具保护性的细胞核的恢复。奇怪的是，用等渗的缓冲区，辅以 NaCl，建议低盐条件可能不最优的线粒体提取更有效地提取线粒体的分数。低渗缓冲考虑汽蚀与低渗缓冲区达到同质化效率和核完整性之间的最佳平衡，成为空我们协议中选择的缓冲区。然而，我们告诫读者没有缓冲区是保证最优同质化的结果。我们的协议应作为一个模板进行优化，并试点测试所需的缓冲区，细胞系的兴趣和其他变量（压力、 缓冲液组成等）必须建立为获得最佳效果。

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 低渗缓冲区中紧接着超速离心法在 350,000 x g 为 1 h。总蛋白从恢复的 S 和 P 的分数进行了分析与标记为 ER、 内涵体和溶酶体、 线粒体、 高尔基体和其他细胞器。所有的跨膜蛋白目前在膜分离完全，包括 Na+/K+ atp 酶和表皮生长因子受体 (EGFR) 从细胞质膜，由内质网、 溶酶体相关的膜蛋白 1 (LAMP1) calnexin从溶酶体，F0-atp 酶从线粒体和 Golgin 97 从跨高尔基网络。不出所料，许多外围膜蛋白与膜松散关联，目前都在不同程度胞浆和膜的组分。显著的例子包括早期体膜抗原 1 (EEA1)，Rab7/9 （晚内小体） 和己糖激酶 1 （外线粒体膜）。这表明此技术在外围蛋白质膜协会实力评价中的实用程序。我们的实验室已利用这种空化技术审查的几个信号分子5，，1213，14膜协会状态。有趣的是，我们发现 calregulin 几乎完全在可溶部分，表明从 ER 膜生成的微粒也已中断期间氮汽蚀和内质网腔中的蛋白质被释放到可溶部分。与此相反的是，空后线粒体的完整性是取决于单元格类型。我们观察到 F1-atp 酶，内在线粒体外周蛋白，部份地在从 NIH 3T3 细胞的 2%~ 35%的人胚肾 293 和白血病细胞的胞浆。这表明，氮在 350 psi 与低渗缓冲区空并不能保证线粒体的完整。尽管三个周期的 500 x g 旋转 PNS 产生空化，并不是所有原子核被都去除了。组蛋白去乙酰化酶 1 (HDAC1) 从核质和核仁，从 fibrillarin 被发现在三七总皂苷和颗粒。HDAC1 颗粒在和不在上清液中的存在表明，三七总皂苷污染与原子核，而不是在空化核中断。这是符合报告，加压到 350 磅炸弹叶核完整10，虽然我们使用低渗缓冲可能使原子核脆弱，正如在后面的章节中讨论。
我们比较同质化效率的氮汽蚀与其他常用的物理中断方法。等量的白血病细胞悬浮在相同的低渗均匀化缓冲区中，遭受不同的中断方法。没有检出损失的样品在 Dounce 同质化和氮空化 （在这两种情况下约 5-10%收集较少的数量）。然而，氮空给蛋白质提取效率最高;针通道和 Dounce 产生只有 60%一样多的蛋白质 (图 3)。奇怪的是，回收一些蛋白质免疫印迹由认定没有显示不同的机械破碎方法的显著差异。然而，如己糖激酶 1 和 RAS 某些外围膜蛋白质产量高于与氮空化制备的样品。这个支持氮气穴现象可能是最佳的同质化调查周边膜蛋白时。回收 HDAC1 蛋白质氮空穴现象相媲美其他方法，这表明原子核保持在所有条件不变。因此，图 3中的数据证明了氮气气穴现象提供了优越的同质化结果。
接下来，我们比较低渗缓冲和相同的缓冲区之间的同质化效率但等渗与蔗糖或氯化钠 (图 4)。蛋白质类似款项被收回从 350 磅/平方英寸的气蚀后均质低渗氯化钠等渗缓冲与缓冲区中的白血病细胞。相比之下，少蛋白回收在细胞破坏在作出与蔗糖等渗的缓冲区中的期票。最个别蛋白质进行免疫印迹适合这种模式。一个例外是相对丰富的三七总皂苷在等渗缓冲区中生成的 fibrillarin。
要确定是否我们的协议可用于调查分区之间可溶性的外周膜蛋白和膜成分，我们比较分区模式之间的旗子标记的国家管制当局野生型和其烯缺陷突变体，C186S （图 5)。野生型国家管制当局在稳定状态，存在于胞液和膜，类似于最外围的膜蛋白，虽然恢复在可溶部分的部分是大于其他 RAS 蛋白14。当国家管制当局剥夺其异戊烯改性时，它丧失了能力与膜相关联，并且成为完全游离。预期的分区模式的国家管制当局证实我们的协议是一个可靠的选项，以便分区外周膜蛋白胞液和膜之间的动力学研究。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56037/56037fig1.jpg"> 
图 1: 流程图总结步骤 2 （蓝色）、 步骤 3 （红色） 和议定书 》 第 4 （黄色） 步。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56037/56037fig1large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56037/56037fig2.jpg"> 
图 2: 蛋白质胞浆和膜组分间的分配。人胚肾 293，NIH 3T3 和白血病细胞受到氮空 (20 分钟，暂停低渗同质化缓冲区中的 ~ 350 psi) 和超速离心法 (1 h ~ 350,000 x g) 议定书 》 中所述。通过免疫印迹法分析了不同馏分中的内源性蛋白水平。三七总皂苷: 受体结合癌抗原表达对单输入单输出细胞 （合），核清、 s: 上清液、 p: 颗粒。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56037/56037fig2large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56037/56037fig3.jpg"> 
图 3: 不同的机械破碎技术的比较。等值的白血病细胞被悬浮在低渗缓冲和受到冷冻-解冻 （3 周期），针通道 （5 经过，28G½ 针），Dounce 同质化 （15 刀，Kontes 2 毫升 Dounce 管与组织研磨杵，清拆0.01-0.06 毫米) 或氮空 （20 分钟 ~ 350 磅/平方英寸）。三七总皂苷中的内源性蛋白的相对水平用免疫印迹法为每个方法进行分析。三七总皂苷的总蛋白浓度进行量化的 BCA 萨y 和归一化氮空化法制备 PNS 的值。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56037/56037fig3large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56037/56037fig4.jpg"> 
图 4: 在氮空蚀过程中使用的低渗、 等渗缓冲区比较。当量的白血病细胞在低渗的缓冲区或低渗缓冲暂停辅以 8.5%的蔗糖或 150 毫米氯化钠，并受到氮空 （20 分钟 ~ 350 磅/平方英寸）。通过为每个缓冲区的免疫印迹分析了三七总皂苷中的内源性蛋白的相对水平。三七总皂苷的总蛋白浓度被量化 BCA 法测定和归 PNS 准备在低渗缓冲区的值。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56037/56037fig4large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56037/56037fig5.jpg"> 
图 5: Farnesylated 国家管制当局划分为 P 和 S 分数。人胚肾 293 细胞瞬时转染指挥的旗子标记的国家管制当局野生型或 C186S 突变体表达质粒。蛋白质的分配进行了如图 2中所述，表明的蛋白质水平进行了免疫印迹。转载与权限 （周，M等人，2016年。最初发表在细胞生物学 》 杂志。https://doi-org.remotexs.ntu.edu.sg/10.1083/jcb.201604061)。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56037/56037fig5large.jpg" target="_blank">请点击这里查看此图的大版本。</a>

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

Nanyang Technological University

Research

JoVE Journal

Biochemistry

15.9K 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

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

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

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

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

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

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological materials. Such approaches have received considerable attention in research on the boundary between living and nonliving matter and have been used to construct artificial cells over the past two decades. In particular, Giant Vesicles (GVs) have often been used as artificial cell membranes. In this paper, we describe the preparation of GVs encapsulating highly packed microspheres as a model of cells containing highly condensed biomolecules. The GVs were prepared by means of a simple water-in-oil emulsion centrifugation method. Specifically, a homogenizer was used to emulsify an aqueous solution containing the materials to be encapsulated and an oil containing dissolved phospholipids, and the resulting emulsion was layered carefully on the surface of another aqueous solution. The layered system was then centrifuged to generate the GVs. This powerful method was used to encapsulate materials ranging from small molecules to microspheres.

Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials.

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

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of several pathogens. Efforts have been dedicated to the cattle tick control to diminish its deleterious effects, with focus on the discovery of vaccine candidates, such as BM86, located on the surface of the tick gut epithelial cells. Current research focuses upon the utilization of cDNA and genomic libraries, to screen for other vaccine candidates. The isolation of tick gut cells constitutes an important advantage in investigating the composition of surface proteins upon the tick gut cells membrane. This paper constitutes a novel and feasible method for the isolation of epithelial cells, from the tick gut contents of semi-engorged R. microplus. This protocol utilizes TCEP and EDTA to release the epithelial cells from the subepithelial support tissues and a discontinuous density centrifugation gradient to separate epithelial cells from other cell types. Cell surface proteins were biotinylated and isolated from the tick gut epithelial cells, using streptavidin-linked magnetic beads allowing for downstream applications in FACS or LC-MS/MS-analysis.

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

A modified density centrifugation gradient-based methodology was utilized to isolate epithelial cells from Rhipicephalus microplus gut tissue. Surface-bound proteins were biotinylated and purified through streptavidin magnetic beads for utilization in downstream applications.

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 ...

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca2+ from mitochondria is important for determining the role of mitochondrial Ca2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca2+ uptake using the Ca2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca2+ in permeabilized cells using the Ca2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca2+. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

Here, we present two protocols for the measurement of mitochondrial Ca2+ influx in isolated mitochondria and cultured cells. For isolated mitochondria, we detail a plate reader-based Ca2+ import assay using the Ca2+ sensitive dye calcium green-5N. For cultured cells, we describe a confocal microscopy method using the Ca2+ dye Rhod-2/AM.

线粒体钙在分离线粒体和培养细胞中的流入分析

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

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

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

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

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

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

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. ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

淀粉样蛋白纤维蛋白的相互作用和脑线粒体的膜渗透

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

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

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.

This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as F&#246;rster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

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

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

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

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.