这项工作得到了澳大利亚研究理事会 （ARC） 发现计划 （DP150101065） 和 ARC 低级最终用户分析集成设备研究中心 （理想） （IH150100028） 的支持。

1. tBLM 电极制备
<ol><li>准备第一层单层涂层<ol>
<li>浸入一个新鲜溅出的金色图案电极显微镜滑动在乙烷溶液中，该溶液由 3 mM 1：9 比例的苯二甲醚- 四乙二醇-OH"垫片"分子（苯二甲醚由四个氧乙二醇垫片组成， 终止与OH组）和苯二硫化物（四乙酰二醇）n=2 C20-植物基"系"分子。这将创建可以固定双层的第一层涂层。 注：金电极通过蒸发100纳米，99.9995%黄金（5n5金）薄膜到定制25毫米x75毫米聚碳酸酯幻灯片20。</li>
		<li>在室温下用第一层孵化电极至少1小时。</li>
		<li>通过浸入大量超过30的纯乙醇来冲洗金电极。</li>
		<li>使用金电极滑梯与第一个单层直接为下一步或存储在一个充满纯乙醇的罐子。</li>
		<li>注意：为了确保第一层的完整性，请尽量减少与幻灯片黄金部分的任何直接接触</li>
	</ol></li>
	<li>组装第一个单层涂层幻灯片<ol>
<li>小心地从容器中取出一个胶质金电极滑梯，使用钳子，确保不会与 tBLM 形成的图案区域接触。 注意：注意识别存放黄金的幻灯片的侧面。</li>
		<li>空气干燥滑梯1 -2分钟，以消除任何残留乙醇。</li>
		<li>将金电极置于干燥表面，确保金电极正确定位，图案金面朝上。</li>
		<li>将透明胶粘层盖从薄层压板中剥离，并放置在 6 个通道上以定义每个井。</li>
		<li>使用压力辊在滑梯和透明粘合层之间释放任何空气，如图1A所示。 注：此步骤所需的时间需要由研究人员优化。在此协议中，时间范围为 2-3 分钟。</li>
		<li>尽快（在 1-2 分钟内）将第二个脂质双层引入组装的第一层涂层电极进行自组装，以避免损坏第一层。</li>
	</ol></li>
	<li>准备第二脂质双层<ol>
<li>在六口井滑梯中的第一口井中加入 6 μL 的 3 mM 脂质。不要让微管尖的边缘接触金表面，金表面会损坏电极上的系绳化学。 注：本工作使用的脂质混合物包括3 mM 70% zwitterionic C20 二苯二甲醚-甘油磷酸酯 （DPEPC） 和 30% C20 二苯二甲醚乙醚脂质 （GDPE） 与 3 mM 胆固醇-PEG-Biotin 混合在 50：1 摩尔比。</li>
		<li>将 6 μL 的脂质混合物引入其他油井，每口油井之间的间隙为 10。</li>
		<li>在室温下将每口井孵育整整2分钟，然后用PBS等缓冲器将脂质混合物交换到电极上。空间的时间添加和缓冲交换10秒分开，所以每口井孵育与脂质正好2分钟每个。</li>
		<li>再洗 3 次，用 50 μL 的 PBS 缓冲器 （pH 7.0） 清洗 3 次。请务必在电极上随时留下 50 μL 的缓冲。不要让电极干燥。 注：用这种方式（溶剂交换方法）将乙醇溶剂与溶剂分离，通过系绳化学快速形成固定在金电极上的单脂双层。</li>
	</ol></li>
	<li>使用电气阻抗光谱 （EIS） 测量测试 tBLM 形成<ol>
<li>将准备好的电极滑动插入交流阻抗光谱仪（例如 Tethapod）。确保光谱仪通过 USB 端口连接到运行软件的计算机。</li>
		<li>打开软件，单击设置并打开硬件。</li>
		<li>设置硬件设置，使用 25 mV 峰值到峰值交流激发。</li>
		<li>将频率设置在 0.1 到 10，000 Hz 之间，每十年设置两个步骤，用于快速阻抗措施按 OK。</li>
		<li>单击设置菜单并打开模型。</li>
		<li>使用等效电路模型，将系绳金电极描述为连续的恒定相位元件，电阻器描述电解质缓冲器和并行电阻电容器网络来描述脂质双层，并按确定。</li>
		<li>按下"开始"按钮，开始实时测量膜电容 （Cm）和膜传导 （Gm）。Cm值的典型 tBLM 应在 12.5 nF 到 15.5 nF 的范围内为 10% 系绳化学21，22。</li>
		<li>运行协议并完成实验后，保存数据。</li>
		<li>用下一口井重复测量。</li>
	</ol></li>
</ol>2.激光照射
<ol><li>实验设置 注：为每个 tBLM 单独设置定制系统。<ol>
<li>在防光箱中执行实验，以最大限度地减少激光危险。</li>
		<li>使用光学表设置实验以减少不必要的振动。</li>
		<li>将阻抗读器（黄金幻灯片连接在哪里）放在 XYZ 舞台上，并提升使其位于激光源的路径上。</li>
		<li>使用粗细聚焦微观齿轮控制激光源的高度，达到适当的精度。</li>
		<li>沿着电极滑梯的纵向轴瞄准激光路径。 注意：始终佩戴合适的激光安全眼镜，保持良好的激光安全规程。</li>
		<li>在开始实验之前，允许选定的调谐激光器稳定下来。 注：实验设置的示意图在图2A中说明。</li>
	</ol>
</li>
	<li>激光和金电极的对齐 注：在开始之前，请始终使用功率计评估激光功率输出，以确保仅向 tBLM 输送非常低的功率。<ol>
<li>调整激光路径或电极的角度，使激光穿过覆盖电极的液体，在金表面均匀可见。</li>
		<li>通过在观察膜传导变化的同时，通过微调提高或降低激光束源来调整每个实验的激光束光位置。</li>
		<li>在没有电导变化时，锁定旋钮以固定激光路径的位置。 注：当激光与底层金电极相互作用时，将产生增加的膜传导值。因此，调整激光路径非常重要，因此不可能进行这种相互作用。</li>
	</ol>
</li>
	<li>样品制备<ol>
<li>准备激光束光对齐（膜传导没有变化），如图2所示，位置3。</li>
		<li>将感兴趣的 GNP（功能化或裸露）添加到 PBS 缓冲器中，当激光关闭时，TBLM 会沉浸其中。</li>
		<li>轻轻混合 TBLM 周围的 PBS 缓冲器三次，小心不要触摸电极。</li>
		<li>在室温下孵育5-10分钟。</li>
		<li>将激光打开以照射样品，使用图2、位置3中正确的对齐激光束光位置。</li>
		<li>使用 GNP 大小、形状和浓度与激光波长的适当组合。 注：设置波长的激光束应耦合到相应的GNP质子共振频率。</li>
		<li>记录连续测量电流（实时测量）。</li>
		<li>执行步骤 2.2.1 - 2.3.7，省略控制实验的 GNP 添加。</li>
	</ol>
</li>
</ol>3.统计数据分析和介绍
<ol><li>将数据导出到电子表格中。</li>
	<li>提取膜传导参数与时间。</li>
	<li>在以正确位置设置激光束灯后和引入 GNP 之前使用记录的数据。</li>
	<li>通过将测量的膜导电度除以基线膜导电性来使数据正常化。 注：这证实了引入辐照GNP引起的膜传导值的相对变化。</li>
	<li>将数据作为时间图（x轴）与规范化膜传导（y轴）。</li>
</ol>4.预测从辐照纳米粒子（热预测模型）中TBLM中产生的局部热量量
<ol><li>根据多布罗夫斯基23日解决辐射转移问题，以计算辐照纳米粒子溶液中的吸收辐射功率。</li>
	<li>通过将吸收辐射产生的热源纳入能量方程来计算热量生成。 注：有关辐照纳米粒子和纳米电极界面的tBLM中热生成数值分析的详细解释，请参阅19。</li>
</ol>

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X., 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=Surface+and+bulk+contributions+to+the+second-order+nonlinear+optical+response+of+a+gold+film.">Surface and bulk contributions to the second-order nonlinear optical response of a gold film.</a> Physical Review B. 80 (23), 233402 (2009).</li></ol>

作者宣布了以下经济利益/个人关系，这可能被视为潜在的竞争利益：布鲁斯·康奈尔教授是外科诊断SDx系绳膜有限公司的科学和技术总监。

该协议描述了使用tBLM模型与平面电极基板结合水平激光对齐设置，使实时电阻抗记录响应激光照射金纳米粒子。此处介绍的 EIS 记录方法构建了一个最少的实验列表，用于记录整个膜的离子电流变化，这与耦合激光和金纳米粒子相互作用产生的热量相对应。本协议中的关键步骤是激光路径向双层脂膜周围的缓冲器的仔细和精确对齐。
<p class="jove_content" fo:keep-together.within...

辐照金纳米材料的高温性能为感染和肿瘤提供了一类新的微创、选择性、有针对性的治疗方法。可以用激光加热的纳米粒子被用来选择性地摧毁患病细胞，并为选择性药物输送提供手段。加热质子纳米粒子光热化现象的后果是对细胞膜的损害。流体脂质双层膜被认为是接受此类治疗的细胞特别脆弱的部位，因为内膜蛋白的变性以及膜损伤也可能导致细胞死亡4，因为许多蛋白质在那里维持细胞膜的离子潜在梯度。虽然确定和监测纳米级传热的能力对研究和应用辐照GNP1、5、6、7、评估和理解GNP与生物膜之间的分子相互作用以及嵌入式GNP在生物组织中的激光诱发加热现象的直接后果至关重要， 尚未完全阐明8。因此，彻底了解辐照GNP的体温过高过程仍然是一个挑战。因此，开发一个模拟细胞自然环境的纳米材料电极接口，可以提供一种手段，对生物系统中辐照金纳米粒子的传热特性进行深入研究。
原生细胞膜的复杂性是理解细胞中辐照GNP相互作用的重大挑战之一。已开发出各种人造膜平台，提供接近简单的生物模仿版本的天然脂膜结构和功能，包括，但不限于，黑色脂膜9，支持平面双层膜10，混合双层膜11，聚合物缓冲脂质双层膜12和系绳双层脂膜13。每个人造脂膜模型在模仿天然脂膜方面具有明显的优点和局限性。
本研究利用tBLM模型，将脂质膜涂层电极作为评估金纳米粒子和脂膜相互作用的传感器。基于tBLM的生物传感器检测方案提供了固有的稳定性和灵敏度13作为系绳膜可以自我修复，不像其他系统（如膜形成的补丁夹或脂质体），其中只有少量的膜损伤导致其崩溃15，16，17，18。此外，由于 tBLM 的尺寸为 mm2，因此背景阻抗的幅度比贴片夹式记录技术低，从而能够记录由于纳米粒子相互作用而导致的基底膜离子通量的变化。因此，本协议可以对比受约束的GNP对膜传导的变化，这些GNP是由其功耗低至135 nW/μm2的激光激发的。
此处介绍的系统为确定设计和开发热疗法所需的精确激光参数、粒子大小、粒子涂层和成分提供了一种敏感且可重复的方法。这对改进新兴的光热疗法至关重要，并为生物系统内的详细传热机制提供了有价值的信息。提交的协议是基于先前公布的工作19。协议大纲如下：第一部分定义 tBLM 形成：第二部分概述了如何构建设置和对齐激发激光源：最后一部分说明了如何从电阻抗光谱数据中提取信息。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>30 nm diameter streptavidin-conjugated gold nanoparticles</td>
			<td>Cytodiagnostics</td>
			<td>AC-30-04-05</td>
			<td>This is a streptavidin-conjugated GNPs product ready for use</td>
		</tr>
		<tr>
			<td>30 nm diameter bare gold nanoparticles</td>
			<td>Sigma-Aldrich</td>
			<td>753629</td>
			<td>This is a bare GNPs product ready for use</td>
		</tr>
		<tr>
			<td>Cholesterol-PEG-Biotin (MW1000)</td>
			<td>NANOCS</td>
			<td>PG2-BNCS-10k</td>
			<td>Dissolved in highly pure ethanol</td>
		</tr>
		<tr>
			<td>C20 Diphytanyl-Glycero-Phosphatidylcholine lipids</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-S1</td>
			<td>1 ml glass vial containing 70% C16 diphytanyl phosphatidylcholine (DPEPC) and 30% C16 diphytanyl glycerol (GDPE) in 99.9% ethanol</td>
		</tr>
		<tr>
			<td>Benzyl-disulfide-tetra-ethyleneglycol-OH</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-S2</td>
			<td>Spacer molecules</td>
		</tr>
		<tr>
			<td>Benzyl-disulfide (tetra-ethyleneglycol) n=2 C20-phytanyl&#160;</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-S2</td>
			<td>Tethered molecules</td>
		</tr>
		<tr>
			<td>532 nm green laser continuous light</td>
			<td>OBIS LS/OBIS CORE LS, China</td>
			<td>ND-1000</td>
			<td>The power of this laser was ~135&#160;mW&#160;</td>
		</tr>
		<tr>
			<td>tethaPod EIS reader</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-R1</td>
			<td>A reader of conductance and capacitance on six channels simultaneously</td>
		</tr>
		<tr>
			<td>tethaPlate cartridge assembly</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-BG</td>
			<td>Materials to attach the slide with electrodes to the flow cell cartridge</td>
		</tr>
		<tr>
			<td>Clamp and slide assembly jig</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-A1</td>
			<td>Materials to attach the slide with electrodes to the flow cell cartridge</td>
		</tr>
		<tr>
			<td>Lipid coated coplanar gold electrodes</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-T10</td>
			<td>Coplanar&#160; gold electrodes are made from 25 mm x 75&#160;mm x&#160;1&#160;mm polycarbonate base substrate with patterned gold arrays layout, then coated with benzyldisulphide, bis-tetraethylene glycol C16 phytanyl half membrane spanning tethers in a tether ratio of 10%&#160;</td>
		</tr>
		<tr>
			<td>tethaQuick software</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-B1</td>
			<td>Software for use with tethaPod to process data and display conductance, impedance and capacitance measurements from the tethaPlate electrodes</td>
		</tr>
		<tr>
			<td>&#160;99.9% Pure ethanol</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>34963</td>
			<td>Absolute,&#160; 99.9%</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>P4417</td>
			<td>pH 7</td>
		</tr>
	</tbody>
</table>

tethered bilayer lipid membranes to monitor heat transfer between gold nanoparticles and lipid membranes

在这里，我们报告了一项协议，使用用金电极组装的系绳双层脂质膜（tBLMs）进行电化学，调查辐照金纳米粒子（GNPs）和双层脂质膜之间的热传递。辐照改性GNP，如链球菌素结合的GNP，嵌入含有目标分子（如生物素）的tBLM中。通过使用这种方法，辐照GNP和具有相关实体的双层脂膜之间的传热过程由水平聚焦激光束进行介导。热预测计算模型用于确认 tBLM 中的电化学诱导导电导变化。在所使用的特定条件下，检测热脉冲需要金纳米粒子与膜表面的特定附着，而未绑定的金纳米粒子则无法引起可测量的反应。该技术作为强大的检测生物传感器，可直接用于设计和开发热疗法策略，从而优化激光参数、颗粒大小、粒子涂层和成分。

可以创建 tBLM 的黄金基板显示在图 1中。实验设置的示意图在图2中提出。
如图1A所示，平面金电极由25毫米×75毫米×1毫米聚碳酸酯基底基板制成，并配有图案黄金阵列。透明胶粘层定义了六个单独的测量室。平面金电极允许激光直接照射到tBLMs膜上。电极阵列的每个井都包含一个圆形工作电极（区域：0.707 cm<s...

Watch this Scientific Journal Video about Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes at JoVE.com

Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

这项工作概述了一项协议，以实现从激光辐照的金纳米粒子到tBLM的热传递的动态、非侵入性监测。该系统结合阻抗光谱，用于实时测量 TBLM 的传导变化，并配有水平聚焦激光束，驱动金纳米粒子照明，用于热生产。

系绳双层脂膜，以监测金纳米粒子和脂膜之间的热传递

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry ...

tethered-bilayer-lipid-membranes-to-monitor-heat-transfer-between

Research

JoVE Journal

Biology

2.6K 次观看.  University of Technology Sydney. 这项工作概述了一项协议，以实现从激光辐照的金纳米粒子到tBLM的热传递的动态、非侵入性监测。该系统结合阻抗光谱，用于实时测量 TBLM 的传导变化，并配有水平聚焦激光束，驱动金纳米粒子照明，用于热生产。

视频: Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

Method for the Isolation of Francisella tularensis Outer Membranes

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other bacterial pathogens, the extremely low infectious dose (&lt;10 CFU), rapid disease progression, and high morbidity and mortality rates suggest that the virulent strains of Francisella encode for novel virulence factors. Surface-exposed molecules, namely outer membrane proteins (OMPs), have been shown to promote bacterial host cell binding, entry, intracellular survival, virulence and immune evasion. The relevance for studying OMPs is further underscored by the fact that they can serve as protective vaccines against a number of bacterial diseases. Whereas OMPs can be extracted from gram-negative bacteria through bulk membrane extraction techniques, including sonication of cells followed by centrifugation and/or detergent extraction, these preparations are often contaminated with periplasmic and/or cytoplasmic (inner) membrane (IM) contaminants. For years, the "gold standard" method for the biochemical and biophysical separation of gram-negative IM and outer membranes (OM) has been to subject bacteria to spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation. Once layered on a sucrose gradient, OMs can be separated from IMs based on the differences in buoyant densities, believed to be predicated largely on the presence of lipopolysaccharide (LPS) in the OM. Here, we describe a rigorous and optimized method to extract, enrich, and isolate F. tularensis outer membranes and their associated OMPs.

Method for the Isolation of Francisella tularensis Outer Membranes

A protocol for separating inner and outer membranes from Francisella tularensis by spheroplasting, osmotic lysis, and sucrose density gradient ultracentrifugation.

隔离方法弗朗西斯tularensis外膜

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other ...

科研

生物学

Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides an indirect way for amphiphiles to modulate protein function and a possible mechanism for "off-target" drug effects. We have previously developed an electrophysiological assay for detecting changes in lipid bilayer properties using linear gramicidin channels as probes 3,12. Gramicidin channels are mini-proteins formed by the transbilayer dimerization of two non-conducting subunits. They are sensitive to changes in their membrane environment, which makes them powerful probes for monitoring changes in lipid bilayer properties as sensed by bilayer spanning proteins. We now demonstrate a fluorescence assay for detecting changes in bilayer properties using the same channels as probes. The assay is based on measuring the time-course of fluorescence quenching from fluorophore-loaded large unilamellar vesicles due to the entry of a quencher through the gramicidin channels. We use the fluorescence indicator/quencher pair 8-aminonaphthalene-1,3,6-trisulfonate (ANTS)/Tl+ that has been successfully used in other fluorescence quenching assays 5,13. Tl+ permeates the lipid bilayer slowly 8 but passes readily through conducting gramicidin channels 1,14. The method is scalable and suitable for both mechanistic studies and high-throughput screening of small molecules for bilayer-perturbing, and potential "off-target", effects. We find that results using this method are in good agreement with previous electrophysiological results 12.

We introduce a fast fluorescence-based assay that monitors the rate of fluorescence quenching as a measure of gramicidin channel activity. The gramicidin channels are used as molecular force transducers to monitor changes in lipid bilayer properties as sensed by bilayer spanning proteins.

短杆菌肽为基础的荧光分析法，确定小分子的修改脂质双层属性的潜在

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter ...

Modeling Biological Membranes with Circuit Boards and Measuring Electrical Signals in Axons: Student Laboratory Exercises

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology used in electrophysiology. The same equipment is used in the membrane model as on live preparations. Some properties of an isolated nerve cord are investigated: nerve action potentials, recruitment of neurons, and responsiveness of the nerve cord to environmental factors.

This is a demonstration of how biological membranes can be understood using electrical models. We also demonstrate procedures for recording action potentials from the ventral nerve cord of the crayfish for student orientated laboratories.

建模生物膜与电路板，并在轴突测量电气信号：学生实验室练习

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

脂质囊泡介导的亲和层析法，使用磁性细胞分选（LIMACS）：一种新的方法来分析蛋白质 - 脂质相互作用

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

重组脂膜的结构和功能研究的钾离子通道

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

采用Caco-2细胞研究脂质运输由肠

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

Isolation and Compositional Analysis of Plant Cuticle Lipid Polyester Monomers

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester of glycerol and modified fatty acids that constitutes the framework of the cuticle that covers aerial plant surfaces. Suberin is a related lipid polyester that is deposited on the cell walls of certain tissues, including the root endodermis and the periderm of tubers, tree bark and roots. These lipid polymers are highly variable in composition among plant species, and often differ among tissues within a single species. Here, we describe a detailed protocol to study the monomer composition of cutin in Arabidopsis thaliana leaves by sodium methoxide (NaOMe)-catalyzed depolymerisation, derivatization, and subsequent gas chromatography-mass spectrometry (GC/MS) analysis. This method can be used to investigate the monomers of insoluble polyesters isolated from whole delipidated plant tissues bearing either cutin or suberin. The method can by applied not only to characterize the composition of lipid polymers in species not previously analyzed, but also as an analytical tool in forward and reverse genetic approaches to assess candidate gene function.

Lipid polyesters constitute the structural components of two cell wall modifications, the plant cuticle and suberin-containing diffusion barriers. In this video, we describe a method to depolymerize cutin from whole delipidated leaves. The method can be applied to investigating mutants compromised in either cutin or suberin biosynthesis.

植物表皮脂质聚酯单体的分离及成分分析

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester ...

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved in humans and are associated with metabolic syndrome or other diseases. Examination of lipid accumulation in this organism can be carried out by fixative dyes or label-free methods. Fixative stains like Nile red and oil red O are inexpensive, reliable ways to quantitatively measure lipid levels and to qualitatively observe lipid distribution across tissues, respectively. Moreover, these stains allow for high-throughput screening of various lipid metabolism genes and pathways. Additionally, their hydrophobic nature facilitates lipid solubility, reduces interaction with surrounding tissues, and prevents dissociation into the solvent. Though these methods are effective at examining general lipid content, they do not provide detailed information about the chemical composition and diversity of lipid deposits. For these purposes, label-free methods such as GC-MS and CARS microscopy are better suited, their costs notwithstanding.

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Nile red staining of fixed Caenorhabditis elegans is a method for quantitative measurement of neutral lipid deposits, while oil red O staining facilitates qualitative assessment of lipid distribution among tissues.

尼罗红与油红 O 染色法测定秀丽线虫的脂质丰度及脂质分布评价

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved ...

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

细胞外通量分析技术在视网膜组织外种中的能量代谢测定

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and diseases, such as obesity and fatty liver. Usually, lipids are stored in lipid droplets, which are the multifunctional lipid storage organelles in cells. Lipid droplets vary in size and number in different tissues and under different conditions. It has been reported that lipid droplets are tightly controlled through regulation of its biogenesis and degradation. In Drosophila melanogaster, the oenocyte is an important tissue for lipid metabolism and has been recently identified as a human liver analogue regarding lipid mobilization in response to stress. However, the mechanisms underlying the regulation of lipid droplet metabolism in oenocytes remain elusive. To solve this problem, it is of utmost importance to develop a reliable and sensitive method to directly visualize lipid droplet dynamic changes in oenocytes during development and under stressful conditions. Taking advantage of the lipophilic BODIPY 493/503, a lipid droplet-specific fluorescent dye, described here is a detailed protocol for the dissection and subsequent lipid droplet staining in the oenocytes of Drosophila larvae in response to starvation. This allows for qualitative analysis of lipid droplet dynamics under various conditions by confocal microscopy. Furthermore, this rapid and highly reproducible method can also be used in genetic screens for indentifying novel genetic factors involving lipid droplet metabolism in oenocytes and other tissues.

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Presented here are detailed methods for the dissection and lipid droplet staining of oenocytes in Drosophila larvae using BODIPY 493/503, a lipid droplet-specific fluorescent dye.

果蝇幼虫的欧诺细胞的解剖和脂滴染色

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and ...