我们感谢布赫兹数字视频制作团队的丹尼尔. 陈, 协助编辑视频。该研究的气孔保护细胞, 蛋白质组学和新陈代谢在陈实验室得到了资助, 美国国家科学基金会 (0818051, 1158000 和 1412547)。文文香港获中国奖学金委员会支持。Qiuying 博士获得中国奖学金委员会和国家自然科学基金 (31570396) 的支持。

1. 种植植物
<ol><li>在灌封混合物中发芽种子。在生长室种植植物在一个光强度的140µmol 光子 m−2 s −1和光周期8小时在22°c 和 16 h 黑暗在18°c 2 星期。</li>
	<li>将类似大小的移栽苗单独放入4英寸直径的花盆中, 并在步骤1.1 所述相同条件下生长3周。 注: 自来水厂每周两次, 以保持土壤湿润。</li>
</ol>2. 制备富气孔
<ol><li>用手术刀把5周大的叶子移开.</li>
	<li>将每片叶子附着在两片透明的透明胶带上, 一条附着在背面 (下部) 一侧, 另一块附着在叶子正面 (上部) 一侧。把带子留在叶子上5秒。</li>
	<li>用食指和拇指分别将两片带子分开, 将背面与人行道、守卫细胞、正面与叶肉细胞分开。</li>
	<li>将果皮从叶背面的60毫升气孔开放缓冲器 (50 毫米氯化钾, 10 毫米 MES, 调整到 pH 6.2 与1米 koh) 在40毫米 x 12 毫米大小的培养皿, 因为他们收集。<ol>
<li>重复上述步骤, 直到收集所有样品皮。</li>
	</ol>
</li>
	<li>在步骤1.1 所述的光条件下, 将含有果皮的培养皿置于生长室中。把果皮放在光下2小时, 完全打开气孔。</li>
	<li>将果皮放入150毫米 x 20 毫米培养皿中, 含有50毫升细胞壁消化酶混合物 (0.7% 纤维素酶 R-10, 0.025% 离析酶 R-10, 0.1% (w/v) polyvinylpyrrolidone-40, 0.25% (w/v) 牛血清白蛋白55% 基本溶液 (0.55 米山梨醇, 0.5 毫米CaCl2, 0.5 毫米氯化镁2, 0.5 毫米抗坏血酸, 10 µM, 2PO4, 5 毫米 4-morpholineethanesulfonic 酸 (MES) 在 pH 5.7 调整与 1 M KOH)。</li>
	<li>在50转20分钟的培养皿中, 在一个相互的振动筛上摇动果皮。</li>
	<li>20分钟后用镊子从酶溶液中取出果皮. 将果皮转移到150毫米 x 20 毫米大小的培养皿中, 含50毫升水。</li>
	<li>使用转移吸管, 冲洗果皮十五年代两次与10毫升的水, 以消除任何残留酶溶液。</li>
	<li>使用镊子将果皮放置在150毫米 x 20 毫米尺寸的培养皿中, 50 毫升的新鲜气孔开启缓冲液在步骤2.4 中使用。在步骤1.1 中所述的光条件下孵育1小时, 以允许气孔恢复。</li>
</ol>3. 脱落酸 (ABA) 治疗及气孔运动测定
<ol><li>在150毫米 x 20 毫米大小的培养皿。在30°c 上孵育富气孔果皮15分钟, 在气孔开启缓冲器的50毫升或气孔开启缓冲器的50毫升内, 以10µM ABA 的最后浓度为适当的处理时间。</li>
	<li>将果皮放在 50 rpm 的振动筛上, 5, 15 和30分钟。每隔一段时间间隔后用镊子去除果皮, 然后按照步骤3.3 中的成像步骤操作。</li>
	<li>采用光镜观察 aba 治疗前的气孔图像和不同的时间点。<ol>
<li>把果皮放在玻璃显微镜滑梯上。</li>
		<li>将幻灯片放在显微镜的舞台上, 调整舞台, 使果皮的图像可以可视化。</li>
		<li>调整在显微镜上的精细和课程重点, 以获得一个清晰的形象的守卫细胞。</li>
		<li>以40X 倍放大倍数的多种气孔图像。</li>
	</ol>
</li>
	<li>使用 ImageJ8测量60气孔光圈。</li>
	<li>在60气孔孔径测量的 p 值 < 0.05 中计算标准误差和意义。</li>
</ol>4. 蛋白质提取和 SDS 分页分离
<ol><li>十五年代用足够的液氮研磨果皮, 用冷冻砂浆和杵盖住果皮。</li>
	<li>每50果皮, 添加3毫升的三饱和苯酚 (pH 8.8) 和3毫升蛋白质提取缓冲 (0.9 米蔗糖, 0.1 米三盐酸, 0.01 米 EDTA, 0.4% 2-巯基乙醇, 10 µL 的1毫米蛋白酶和磷酸酶抑制剂) 与一个吸管的砂浆, 然后研磨的果皮5此外在通风罩分钟。</li>
	<li>使用吸管和果皮用金属镊子将萃取物转移到欧克瑞奇离心管上, 并在 50 rpm 1 小时的振动筛上搅动4摄氏度。</li>
	<li>从欧克瑞奇离心管中取出果皮, 离心萃取物在 5000 x g 处10分钟。然后用吸管将顶酚相转移到新的清洁微离心管上。</li>
	<li>在100% 甲醇中加入5卷冰冷0.1 米醋酸铵, 沉淀苯酚提取蛋白。漩涡简要地和孵化隔夜在-20 °c。</li>
	<li>离心机在 2万 x g 在4°c 为20分钟, 醒酒在上清液慢慢地倾吐它从管子并且保留蛋白质药丸在管子里。</li>
	<li>用0.1 米醋酸铵在甲醇中两次洗涤蛋白颗粒, 然后两次用80% 丙酮, 一次用100% 冷丙酮。 注: 通过将10毫升的指定试剂添加到蛋白质管中进行洗涤。在 50 rpm 5 分钟的振动筛上搅拌, 其次为 1.5万 x g 的离心, 4 摄氏度为10分钟。然后将试剂倒入试管中, 只保留蛋白颗粒。</li>
	<li>加入1毫升100% 冷丙酮的颗粒, 并通过缓慢吹打的向上和向下重新暂停。</li>
	<li>通过吸管转移蛋白质悬浮液到2毫升微离心管, 然后离心机在 2万 x g 在4°c 5 分钟。</li>
	<li>将丙酮醒酒从管中取出, 在通风罩中干燥10分钟。</li>
	<li>用200µL 溶解缓冲液 (8 米尿素、0.5% SDS、30毫米三盐酸盐在 pH 值 8.5) 和涡旋为 30 x g 在2万摄氏度为15度, 在新管中收集上清, 将蛋白质溶解在微离心管中。</li>
	<li>根据蛋白质量化协议9, 量化蛋白质浓度。</li>
	<li>在10号议定书之后使用蛋白质样品进行蛋白质分离。</li>
</ol>

<ol>
	<li>Acharya, B., Assmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormone+interactions+in+stomatal+function.">Hormone interactions in stomatal function.</a> Plant Molecular Biology. 69 (4), 451-462 (2009).</li><li>Schroeder, J. I., Allen, G. J., Hugouvieux, V., Kwak, J. M., Waner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guard+cell+signal+transduction.">Guard cell signal transduction.</a> Annual Review of Plant Physiology and Plant Molecular Biology. 52, 627-658 (2001).</li><li>Obulareddy, N., Panchal, S., Melotto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guard+Cell+Purification+and+RNA+Isolation+Suitable+for+High-Throughput+Transcriptional+Analysis+of+Cell-Type+Responses+to+Biotic+Stresses.">Guard Cell Purification and RNA Isolation Suitable for High-Throughput Transcriptional Analysis of Cell-Type Responses to Biotic Stresses.</a> Molecular Plant-Microbe Interactions. 26 (8), 844-849 (2013).</li><li>Pandey, S., Wang, X. Q., Coursol, S. A., Assmann, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+applications+of+Arabidopsis+thaliana+guard+cell+protoplasts.">Preparation and applications of Arabidopsis thaliana guard cell protoplasts.</a> New Phytologist. 153 (3), 517-526 (2002).</li><li>Schroeder, J. I., Raschke, K., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage+Dependence+Of+K++Channels+In+Guard-Cell+Protoplasts.">Voltage Dependence Of K+ Channels In Guard-Cell Protoplasts.</a> Proceedings of the National Academy of Sciences of the United States of America. 84 (12), 4108-4112 (1987).</li><li>Wu, F. H., 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=Tape-Arabidopsis+Sandwich+-+a+simpler+Arabidopsis+protoplast+isolation+method.">Tape-Arabidopsis Sandwich - a simpler Arabidopsis protoplast isolation method.</a> Plant Methods. 5, 10 (2009).</li><li>Svozil, J., Gruissem, W., Baerenfaller, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteasome+targeting+of+proteins+in+Arabidopsis+leaf+mesophyll,+epidermal+and+vascular+tissues.">Proteasome targeting of proteins in Arabidopsis leaf mesophyll, epidermal and vascular tissues.</a> Frontiers in Plant Science. 6, (2015).</li><li>Schneider, C., Rasband, W., Eliceiri, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li><li>Agnew, B., Murray, D., Patton, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+solid-phase+fluorescence-based+protein+assay+for+quantitation+of+protein+electrophoresis+samples+containing+detergents,+chaotropes,+dyes,+and+reducing+agents.">A rapid solid-phase fluorescence-based protein assay for quantitation of protein electrophoresis samples containing detergents, chaotropes, dyes, and reducing agents.</a> Electrophoresis. 25 (15), 2478-2485 (2004).</li><li>He, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laemmli-SDS-PAGE.">Laemmli-SDS-PAGE.</a> Bio-protocol. Bio101 (80), (2011).</li><li>Thompson, A., 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=Tandem+mass+tags:+A+novel+quantification+strategy+for+comparative+analysis+of+complex+protein+mixtures+by+MS/MS+(vol+75,+pg+1895).">Tandem mass tags: A novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS (vol 75, pg 1895).</a> Analytical Chemistry. 78 (12), 4235-4235 (2006).</li><li>Wiese, S., Reidegeld, K. A., Meyer, H. E., Warscheid, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+labeling+by+iTRAQ:+A+new+tool+for+quantitative+mass+spectrometry+in+proteome+research.">Protein labeling by iTRAQ: A new tool for quantitative mass spectrometry in proteome research.</a> Proteomics. 7 (3), 340-350 (2007).</li></ol>

没有宣布利益冲突。

保护细胞是研究植物信号转导机制的模型系统, 重要的是要确保研究中使用的样品的制备最适合回答生物学问题。尽管植物研究界对保护细胞的兴趣越来越浓厚, 但对于如何制备气孔保护细胞, 使气孔运动和生理以及分子变化在一个研究中都没有普遍的方法系统。隔离它们的挑战在很大程度上归因于小规模、低丰度和独特的结构, 这可以使它们或多或少难以从整个树叶中移除。
...

气孔在植物的整体生理和健身中起着重要的作用。这些微小的植物特定的结构是由两个专门的表皮细胞组成的, 称为守卫细胞, 在叶的背面最丰富的发现。气孔是在植物和大气之间交换气体和通过蒸腾控制水分损失所必需的。保护细胞通过不同外部 (例如, 光, 湿度, 病原体接触, 二氧化碳水平) 和内部 (e., 内源激素) 的整合来调节气孔孔径,刺激 1,2。在过去的20年中, 这种细胞类型已经成为研究植物细胞信号过程的模型系统。这些细胞构成了叶中总细胞的一小部分;因此, 以单细胞的方式研究其独特的细胞、生物化学和分子特性, 就有必要将它们与其他叶细胞类型分离。在过去, 守卫细胞研究经常介入了卫兵细胞原生质体的准备 (GCP)3,4,5。这通常需要使用大量的细胞壁降解酶和或通过混合的机械干扰, 并证明是昂贵和耗时, 因为它通常需要很多小时来准备一个 GCP 样品, 往往是很少的产量。更重要的是, 由于保护细胞作为完全对调节气孔孔径, 使用 GCP 可以被视为一个人工系统, 研究保护细胞信号。
在这里, 我们开发了一种方法来制备气孔, 其中完整的保护细胞丰富和保持生理反应的刺激。这种方法的灵感来自于分离叶肉细胞原生质体6,7的方法。在我们的方法, 气孔是准备使用透明的透明胶带, 首先分离的大部分叶肉细胞的正面层的叶子从背面层包含路面和守卫细胞。这是通过贴一块胶带到叶子的两侧, 并剥离它们分开。在气孔开放的缓冲区中, 允许从背面层的细胞恢复到光下。后来, 其余的路面细胞被删除使用少量的细胞壁降解酶, 留下保护细胞, 丰富的磁带。
在这种简单的方法中, 气孔保护细胞既可行又反应灵敏, 可以快速有效地制备, 花不到一小时就能从50皮中获得丰富的气孔保护细胞, 成本最低。这里的方法对植物细胞的损伤比以前的原生质体制备方法少。此外, 从这种方法收集的材料产生理想的蛋白质量。最重要的是, 由于叶子不受混合或长时间的消化, 并且保护细胞保持不变, 研究结果将更接近于保护细胞的自然生物学。利用这种方法, 气孔运动可以与分子水平的变化实时相关。因此, 从研究中获得的知识使用这种制备方法, 对于更全面地了解防护细胞信号是非常重要的。
我们用植物拟南芥作为模型;但该协议可应用于其他植物中的气孔保护细胞的富集, 如甘蓝型油菜的消化时间小改性。作为概念的证明, 我们已经证明, 通过这种方法丰富的气孔保护细胞是可行的, 并响应的刺激, 如荧光素二乙酸 (FDA) 的研究, 并利用植物激素脱落酸 (ABA), 分别。此外, 我们已经证明, 高质量的蛋白质可以从这些守卫细胞分离。在这里, 我们描述了这个过程的详细协议。

<table border="0" cellpadding="0" cellspacing="0" style="width:889px;" width="889">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Stainless Steel Surgical Scalpel</td>
			<td style="width:297px;">Feather&#160;</td>
			<td style="width:119px;">&#160;NC9999403</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Scotch Tape (3M)</td>
			<td style="width:297px;">ULINE</td>
			<td style="width:119px;">S-9781</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Petri Dish</td>
			<td style="width:297px;">Sigma-Aldrich</td>
			<td>BR455701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cellulase (Onozuka R-10)&#160;</td>
			<td>Yakult Pharmaceutical Industry Co., Ltd.</td>
			<td>21560003-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Macerozyme R-10</td>
			<td>Yakult Pharmaceutical Industry Co., Ltd.</td>
			<td>21560003-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">DM6000B Microscope&#160;</td>
			<td>Leica Microsystems</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease and Phosphatase Inhibitor Cocktails</td>
			<td style="width:297px;">Thermo Fisher Scientific&#160;</td>
			<td>&#160;PI78443</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Oak Ridge High-Speed PPCO Centrifuge Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>3119-0030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EZQ Protein Quantitation Kit</td>
			<td style="width:297px;">Thermo Fisher Scientific</td>
			<td>R33200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescein Diacetate (FDA)</td>
			<td style="width:297px;">Thermo Fisher Scientific</td>
			<td>F1303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Abscisic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Metro Mix 500</td>
			<td style="width:297px;">BWI Companies&#160;</td>
			<td style="width:119px;">TX-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Laemmli Sample Buffer</td>
			<td style="width:297px;">Bio-Rad</td>
			<td style="width:119px;">161-0737</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Bio-Safe Comassie (G-250)</td>
			<td style="width:297px;">Bio-Rad</td>
			<td style="width:119px;">161-0786</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Microcentrifuge Tube (2mL)</td>
			<td style="width:297px;">USA Scientific, Inc.</td>
			<td style="width:119px;">1620-2700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">ImageJ software</td>
			<td style="width:297px;">National Institute of Health</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Tweezers&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F4017-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">12% Mini-PROTEAN TGX Precast Gel</td>
			<td style="width:297px;">Bio-Rad</td>
			<td style="width:119px;">456-1043</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope slides</td>
			<td style="width:297px;">Fisherbrand</td>
			<td>12-550-A3</td>
			<td></td>
		</tr>
	</tbody>
</table>

stomata tape-peel: an improved method for guard cell sample preparation

保护细胞的研究对于了解这种细胞类型对植物整体功能的特殊贡献是至关重要的。然而, 通常很难分离, 因此研究它们往往会带来挑战。
本研究建立了气孔保护细胞的富集方法。该协议利用透明胶带分离出保护细胞并从细胞中提取蛋白质。该方法在隔离过程中提高了保护细胞的完整性和产量, 为细胞信号过程的研究以及它们与气孔运动表型的相关性提供了可靠的样本。在这种方法中, 植物叶被分成两部分的胶带和剥离分开, 以消除背面的一侧。本协议适用于拟南芥叶组织分离保护细胞。用荧光素双乙酸 (FDA) 对细胞进行治疗, 以确定活性和脱落酸 (aba), 以评估 aba 对气孔运动的反应。最后, 该协议对制备富含气孔保护细胞和分离蛋白质具有重要意义。这里获得的细胞和蛋白质的质量使生理学和其他生物研究得以实现。

图 1显示了拟南芥保护细胞在叶肉和表皮细胞消化前后的代表性图像。使用 FDA 测量酶活性和细胞膜完整性 (图 1B和1D) 可以观察到叶肉和表皮细胞去除前后的保护细胞活力。图 2说明拟南芥的气孔运动对 ABA 的治疗反应。与 ABA 治疗30分钟后气孔孔径减少50% 以上。图 2A是气?...

Watch this Scientific Journal Video about Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation at JoVE.com

Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation

本协议描述了一种制备富含气孔保护细胞的方法, 用于生理学和其他生物研究。

气孔带皮: 一种改进的防护细胞样品制备方法

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is ...

stomata-tape-peel-an-improved-method-for-guard-cell-sample-preparation

Research

JoVE Journal

Biology

14.9K 次观看.  University of Florida. 本协议描述了一种制备富含气孔保护细胞的方法, 用于生理学和其他生物研究。

视频: Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI样品制备：超薄层的方法

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

科研

生物学

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

大会，加载和分析超速离心机样品池对齐

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

免疫印迹检测样品制备

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

Preparation of Quality Inositol Pyrophosphates

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells are inositol pentakisphosphate (IP5) and inositol hexakisphosphate (phytic acid or IP6). IP5 and IP6 are the precursors of inositol pyrophosphate molecules that contain one or more pyrophosphate bonds1. Phosphorylation of IP6 generates diphoshoinositolpentakisphosphate (IP7 or PP-IP5) and bisdiphoshoinositoltetrakisphosphate (IP8 or (PP)2-IP4). Inositol pyrophosphates have been isolated from all eukaryotic organisms so far studied. In addition, the two distinct classes of enzymes responsible for inositol pyrophosphate synthesis are highly conserved throughout evolution2-4.
The IP6 kinases (IP6Ks) posses an enormous catalytic flexibility, converting IP5 and IP6 to PP-IP4 and IP7 respectively and subsequently, by using these products as substrates, promote the generation of more complex molecules5,6. Recently, a second class of pyrophosphate generating enzymes was identified in the form of the yeast protein VIP1 (also referred as PP-IP5K), which is able to convert IP6 to IP7 and IP87,8.
Inositol pyrophosphates regulate many disparate cellular processes such as insulin secretion9, telomere length10,11, chemotaxis12, vesicular trafficking13, phosphate homeostasis14 and HIV-1 gag release15. Two mechanisms of actions have been proposed for this class of molecules. They can affect cellular function by allosterically interacting with specific proteins like AKT16. Alternatively, the pyrophosphate group can donate a phosphate to pre-phosphorylated proteins17. The enormous potential of this research field is hampered by the absence of a commercial source of inositol pyrophosphates, which is preventing many scientists from studying these molecules and this new post-translational modification. The methods currently available to isolate inositol pyrophosphates require sophisticated chromatographic apparatus18,19. These procedures use acidic conditions that might lead to inositol pyrophosphate degradation20 and thus to poor recovery. Furthermore, the cumbersome post-column desalting procedures restrict their use to specialized laboratories.
In this study we describe an undemanding method for the generation, isolation and purification of the products of the IP6-kinase and PP-IP5-kinases reactions. This method was possible by the ability of polyacrylamide gel electrophoresis (PAGE) to resolve highly phosphorylated inositol polyphosphates20. Following IP6K1 and PP-IP5K enzymatic reactions using IP6 as the substrate, PAGE was used to separate the generated inositol pyrophosphates that were subsequently eluted in water.

Inositol pyrophosphates play an important role in human pathologies such cancer, diabetes and obesity; however, the exact mechanism of action is a matter of dispute. The lack of commercially available inositol pyrophosphates renders detailed studies problematic. Here we describe a simple protocol to produce and isolate milligrams of inositol pyrophosphates.

编制质量肌醇Pyrophosphates

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

在对飞行性能的准确，快速测定方法的改进<em&gt;果蝇</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

单病毒原子力显微镜和超分辨率荧光成像的样品制备

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current literature describes several different methodological approaches to obtain images of intestinal tissues for data validation. In this paper, we delineate a common protocol relating to the derivation and processing of mouse intestinal tissues. Proper fixation of intestinal tissues and Swiss-roll techniques that enhance intestinal epithelial morphology are discussed. Postresection processing and reorientation of embedded intestinal tissues are critical in obtaining paraffin-embedded blocks that display intact intestinal structural features after sectioning. The Swiss-rolling technique helps in histological assessment of the complete intestinal or colonic sections examined. An ability to differentiate intestinal structural features can be vital in quantitative measurements of intestinal inflammation and tumorigenesis along the entire length. Finally, paraffin-embedded sections are ideal for robust processing using both immunohistochemical and immunofluorescent detection methods. Nonfluorescent immunohistochemical sections provide a vibrant image of the tissue detailing different cellular structural features but do not provide flexibility for intracellular co-localization experiments. Multiple fluorescent channels can be appropriately utilized with immunofluorescent detection for co-localization experiments, lending support to mechanistic studies.

Accurate identification and location of epithelial cells along the intestinal mucosal lining are essential to define different cell lineages. Proper imaging of intestinal tissues is crucial for identification of protein expression patterns with maximum resolution. This study aims to delineate the optimal methods and conditions for processing mouse intestinal tissues.

瑞士改进轧制技术肠组织准备免疫组化和免疫分析

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current ...

Sample Preparation for Mass Cytometry Analysis

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.

This article describes the collection and processing of samples for mass cytometry analysis.

样品制备质谱术分析

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

对于样品制备与TEM分析低细胞数量的另一种方法

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

混合式切割:用于顽拗植物组织样品的改进切法

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...