原子力显微镜成像和支持的脂质双层力谱
Summary
We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.
Abstract
Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force.
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.
Introduction
原子力显微镜（AFM）由扫描用悬臂具有非常尖锐的端部1产生在样品的一个区域的表面的图像。悬臂的运动探测样品的表面拓扑结构。 AFM已经广泛应用于生物分子-包括蛋白质，DNA，和膜，由于它的多功能性在分析中液体2-5在空气中或接近天然状态固定的样品。
除了 其在纳米范围内的高分辨率成像能力，原子力显微镜悬臂充当弹簧探测的相互作用力（粘附力和斥力）和样品5,6的机械性能。这就是所谓的力谱。在这种模式下，探针先靠近样品和施加在其上的力，然后缩回，直到它失去接触与样品（ 图1A）。所产生的曲线显示力作为距离悬臂的两个应用程序的函数蟑螂和回缩。几个属性，包括弹性模量测量材料的硬度和粘合力可以得到。
支持的脂质双层的生物模型膜躺在了坚实的支撑顶部-通常是云母，硼硅玻璃，熔融石英，或氧化硅7。他们使用的是一样的囊泡沉积的各种技术制备，朗缪尔-布洛杰特法和旋涂8,9。原子力显微镜成像已被用来按照下列支持双层10的形成，并探测由不同的组合物11-15的膜形成不同的结构。
执行力谱上支持双层结果的方法曲线的峰值。这表明峰值刺穿双层所需的力，被称为突破力。该双层厚度，也可以使用力曲线6测量。双层典型的突破力1-50之间范围NN 6。这些特性取决于脂质填料（液体或凝胶相）和结构（酰基链长和不饱和度），并通过膜活性剂16改变。破裂背后的理论已经解释17等实验参数，如悬臂柔软度，刀尖半径和进近速度也影响了突破力15,16,18。力光谱已被用来分析不同的脂质相11,19，组成依赖变化12,20，以及其它生物分子的效果，例如，肽的性质，在膜21的稳定性。
支持双层的平面取向是有利的原子力显微镜和其他方法，例如表面等离子共振22和荧光显微镜11,19组合以更好地表征结构和膜的特性。
这种详细的原始视频COL旨在利用囊泡沉积制备支持的脂质双层和他们AFM和力谱分析。虽然可以使用各种尺寸的囊泡以制备双层，该协议的重点是小的和大单层囊泡。支持双层相分离成液体下令（L O）和液体紊乱（L D）阶段即进行了表征11,15。 2：1的比例将膜在2由二油酰 – 磷脂酰胆碱（DOPC），鞘磷脂（SM）和胆固醇（CHOL）的。该组合物车型脂筏，这是建议的行为作为平台蛋白质运输和分拣，细胞信号和其他细胞的过程23,24重要。
Protocol
1.准备支持的脂质双层（SLB）11,12,21 脂质混合物和多层囊泡悬浮液的制备事先准备以下的缓冲区。 制备PBS缓冲液以2.7毫米氯化钾，浓度为1.5mM KH 2 PO 4，8毫的Na 2 HPO 4，和137 mM氯化钠，pH值7.2。 制备SLB（支持的脂质双层）缓冲液，在150 mM氯化钠，的10mM HEPES，pH 7.4中的浓度。 制备的1M的CaCl 2的溶液中。 ?…
Representative Results
DOPC构成支持的脂质双层：缅澈（2：2：1）进行成像的原子力显微镜（ 图2的AC）。由于脂质成分，SM /澈丰富1。·和DOPC丰富l D同时相观察。从原子力显微镜成像高度轮廓可以提供在膜结构的重要信息。通过查看高度轮廓，该双层厚度可在缺陷在膜中（ 图2B），或的Lø/ L D-相之间的高度差存在待测量可以提供。此外，AFM允许代表这些阶段具体选?…
Discussion
的SLBs DOPC组成：缅澈（2：2：1）中诱导氯化钙囊泡吸附和破裂后形成在云母。这种脂质成分分离为L- D和LØ阶段。的1。·相位富含鞘磷脂和胆固醇，小于流体/更粘（ 图1A）比l D同时相11。 1。·了由L D相分离的表现高于周围（ 图1B，C）升高为圆形结构。该平台是由l D同时相包围1。·阶段。膜缺陷（或…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项工作是由马普学会，图宾根大学的德国癌症研究中心，以及德国联邦联邦教育与研究部（批准号：0312040）的支持。
我们感谢爱德华·赫尔曼帮助我们自动力曲线数据和雅各布Suckale博士仔细阅读本手稿的分析。
Materials
| 1,2-dioleoyl-sn-glycero-3-phosphocholine | Avanti Polar Lipids, Inc. | 850375P | Comes as lyophilized powder in sealed vials. Dissolve all powder in chloroform upon opening. Store extra as dried lipid films, under inert atmosphere, at -20°C. For more information on storage and handling visit http://www.avantilipids.com/index.php?option=com_content&view=article&id=1679&Itemid=398 |
| Sphingomyelin (Brain, Porcine) | Avanti Polar Lipids, Inc. | 860062P | Comes as lyophilized powder in large sealed plastic containers. Dissolve a spatula point of powder powder in chloroform upon opening. Store extra as dried lipid films, under inert atmosphere, at -20°C. For more information on storage and handling visit http://www.avantilipids.com/index.php?option=com_content&view=article&id=1679&Itemid=398 |
| Cholesterol | Avanti Polar Lipids, Inc. | 700000P | Comes as lyophilized powder in large sealed plastic containers. Dissolve a spatula point of powder powder in chloroform upon opening. Store extra as dried lipid films, under inert atmosphere, at -20°C. For more information on storage and handling visit http://www.avantilipids.com/index.php?option=com_content&view=article&id=1679&Itemid=398 |
| Sodium chloride (NaCl), 99.8% | Carl Roth GmbH + Co. KG | 9265.1 | |
| Potassium chloride (KCl), 88% | Sigma | P9541 | |
| Sodium hydrogenphosphate (Na2HPO4), >99% | AppliChem GmbH | A1046 | |
| Potassium dihydrogenphosphate (KH2PO4), 99% | Carl Roth GmbH + Co. KG | 3904.1 | |
| Calcium chloride dihydrate (CaCl2), molecular biology grade | AppliChem GmbH | A4689 | |
| HEPES, molecular biology grade | AppliChem GmbH | A3724 | |
| Glass coverslip, 24×60 mm, 1mm thickness | Duran Group | 2355036 | |
| Mica blocks | NSC Mica Exports Ltd. | These are mica pieces at least 1 sq. Inches in area and thickness randing from 0.006 inches to 0.016 inches. They are cut to a specific size by the company for shipping. Small mica discs can be punched from the mica blocks using the punch and die set. Always handle mica with gloves or tweezers. | |
| Punch and Die Set | Precision Brand Products, Inc | 40105 | |
| Optical Adhesive | Norland Products, Inc. | NOA 88 | Liquid adhesive that hardens when cured under long wavelength UV light. |
| Laboratory Equipment Grease | Borer Chemie AG | Glisseal N | |
| Liposome Extruder | Avestin | LiposoFast-Basic | As an alternative one can also look at offers from Northern Lipids, Inc. |
| Adhesive Tape | 3M | Scotch(R) Magic (TM) Tape 810 (1-inch) | |
| Bath Sonicator | Bandelin Sonorex Digitec | DT-31 | No heating, Frequency: 35 kHz, Ultrasonic Peak Output: 160 W, HF Power: 40 W. http://www.bandelin.com/datenblaetter/dt/DT_31_H_1798d_DE_GB_FR_BANDELIN.pdf |
| Silicon Nitride AFM Cantilever | Bruker AFM Probes | DNP-10 | Each cantilever has four tips and their nominal tip radius is 20 nm (with possible maximum at 60 nm). Based on the specifications, we use tip D with resonance frequency of 18 kHz, and nominal spring constant of 0.06 N/m. |
| AFM | JPK | JPK Nanowizard II mounted on Zeiss Axiovert 200 |
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Citar este artículo
Unsay, J. D., Cosentino, K., García-Sáez, A. J. Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers. J. Vis. Exp. (101), e52867, doi:10.3791/52867 (2015).