Name: reconstitution of a transmembrane protein, the voltage-gated ion channel, kvap, into giant unilamellar vesicles for microscopy and patch clamp studies
Uploaded: 2015-01-22T13:00:00
Description: Watch this Scientific Journal Video about Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies at JoVE

We thank Susanne Fenz for discussing the possibility of reconstituting proteins by agarose swelling, Feng Ching Tsai for current measurements, and present and former members of the Bassereau group for support and assistance. The project was funded by the Agence Nationale de la Recherche (grant BLAN-0057-01), by the European Commission (NoE SoftComp), by the Universit&#233; Pierre et Marie Curie (grant from the FED21, Dynamique des Syst&#232;mes Complexes). M.G. was supported by an Institut Curie International PhD Fellowship, S.A. by a fellowship from the Fondation pour la Recherche M&#233;dicale, G.E.S.T. by a Marie Curie Incoming International Fellowship from the European Commission and a grant from the Universit&#233; Pierre et Marie Curie. The publication fees were covered by the Labex &#8216;CelTisPhyBio&#8217; (ANR-11-LABX0038).

1.溶液制备<ol><li>制备5ml含5mM的氯化钾，1mM的HEPES（pH 7.4）中或TRIS（pH为7.5），和2mM海藻糖&#39;的SUV缓冲器“的。过滤用0.2微米的针筒式过滤器的缓冲器和分成可存储在-20℃1ml等分。 注：其他详细信息的试剂和仪器的材料清单给出。 </li><li>准备40毫升GUV“成长缓冲”，这将填补室内GUV在电影补液。对于一个“低盐”的增长，结合的5mM的KCl，1mM的HEPES（pH 7.4）中或1mM的TRIS（pH为7.5）和〜400毫蔗?...

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仿生模型系统用于研究蛋白质和膜的性能和相互作用的一个重要工具。相比像类脂膜或支持的脂质膜的其他重组系统，GUV基础的系统本数的机会，包括相当大的控制的膜组合物，紧张和几何形状，以及作为真正的无油。然而，掺入跨膜蛋白，如KvAP成GUVs需要常规的协议为脂质仅GUVs显著适应。这里介绍的electroformation协议是以前的特点，并用于研究在弯曲膜2,4,35膜蛋白的分布和动态变化的生?...

当研究支配生命系统的物理原理，自下而上途径允许一个实验，以控制系统的组成和不容易在基于细胞的系统1操作的其他参数。为基于膜的工艺，巨单层囊泡（GUVs，直径〜1-100微米）已被证明是一种非常有用的仿生系统2 - 7，因为它们非常适合于显微镜研究和显微8 - 10。虽然有许多不同的协议来产生GUVs，最分为两类-基乳液方法的基础上再水化脂质膜13 11,12和技术- 16。在乳化液为基础的方法中，GUV膜的内，外小叶被依次从脂质单层在水/油界面组装。这种方法适用于封装可溶性蛋白与在GUVs，和用于形成GUVs不对称小叶脂质组合物。然而，从乳液形成GUVs可以保留的痕量溶剂是改变膜的机械特性17，并且该方法不特别适合于跨膜蛋白重建。 膜补液方法依赖于以下事实干燥（脱水）导致许多脂类混合物以形成膜的多片层堆栈。如果此堆栈，然后放置在接触含水缓冲液，在叠层膜会移动它们之间，并在该叠层的表面分开为溶剂流动，个别膜可以分离，以形成GUVs 13,18（以及一个真正的动物园其它脂质对象）。然而，即使是对于最佳缓冲液和脂质组合物，这种典型的“自发溶胀”的方法具有相对低的无缺陷GUVs收率。一种广泛使用的方法，以提高无缺陷GUVs收率“electroformation221 ;,在其中交变电流（AC）场期间膜补液施加。而该机制仍然知之甚少，“electroformation”可以给予壮观GUV产率（&gt;在有利的情况下，90％）为低盐浓度缓冲液（&lt;5毫米）14,19，并且甚至可以在生理缓冲液的工作（约100毫米）使用较高的频率（500赫兹与10赫兹）的交流场和铂电极15。另一种方法，以促进无缺陷GUVs收率是“凝胶辅助肿胀”，其中的脂质溶液沉积在聚合物凝胶基质，而不是被动用于古典“自发溶胀（ 例如 ，玻璃，聚四氟乙烯）基底“。当所得到的脂质/凝胶膜再水化，GUVs能迅速形成，即使对于生理缓冲器16,20。 所有这些方法都可以产生可用于研究膜相关的现象，如脂质仅GUVs可溶性蛋白和膜之间的相互作用。然而，掺入跨膜蛋白进入GUVs，需要显著修改，以确保该蛋白质保持在功能状态在整个重构过程。而在有机溶剂（ 例如，氯仿，环己烷）脂质的解决方案非常适合用于制造脂质膜，跨膜蛋白是典型地仅当稳定它们的疏水跨膜结构域被嵌入脂质双层，或由洗涤剂胶束包围（ 例如，在蛋白质纯化）。因此，对于一重建的起始材料通常是由洗涤剂去除在形成的天然的膜，在洗涤剂溶液中纯化的蛋白质，或小单层含蛋白囊泡（PROTEO-越野车）和/或多层状囊泡（PROTEO-MLV的）存在脂质。大多数将这些膜蛋白进入GUVs方法分为三类。 直接InsertioN：跨膜蛋白悬浮在洗涤剂与预成形的，脂质-只，轻度清洁剂溶解的GUVs混合，并且将洗涤剂然后使用biobeads 21除去。而概念简单，这种方法需要的洗涤剂浓度的精确控制，因为太高的洗涤剂浓度可以溶解而过低的浓度可导致蛋白质折叠或聚集的GUVs。 GUV / PROTEO-SUV的融合：蛋白质在PROTEO-SUV的组合与预成形的，脂质-仅GUVs和融合促进了与特殊膜融合肽22或21的洗涤剂。典型地融合的程度有限，导致GUVs具有低蛋白质浓度。 脱水/再水化：包含蛋白质 - 类脂膜通过PROTEO-SUV的部分脱水（或PROTEO-MLV）溶液和GUVs形成然后生长作为用于纯脂质膜。明显的挑战是局部dehydrati期间保护蛋白质关于步骤23中 ，但该方法已被成功地用于重组跨膜蛋白，如细 ​​菌视紫红质，钙ATP酶，整合和VDAC成GUVs 7,23 - 25。 本文介绍的脱水/再水化协议，使含有该电压-门控钾通道，KvAP，从超嗜热古菌， 嗜pernix GUVs。KvAP具有高度的同源性，以真核电压依赖性钾通道26和一个公知的晶体结构27 ，使其成为一个很好的模型来研究电压门控的机制。生产PROTEO-SUV的已详细描述以前，不是本教程26,28,29的一部分。重要的是，KvAP PROTEO-SUV的不必要产生的每个GUV制剂，因为它们可以被存储在小（ 例如，10微升）等分在-80℃下延长的时间段（&gt; 1年）。 Electroformation或凝胶辅助溶胀然后可用于从KvAP PROTEO-SUV的生长GUVs（或PROTEO-MLV的）。 为electroformation协议的关键步骤被示于图1。含有蛋白质的SUV的溶液的液滴沉积在铂丝（ 图2中示出）。在SUV的悬挂部分脱水导致通过的SUV的融合，形成脂质蛋白膜。期间补液，一个交流电场施加到电极上，以协助脂质层脱层并形成GUVs。 10赫兹场效果很好时，使用“低盐”（&lt;5毫米）补液缓冲区28和GUVs需要几个小时才能成长。与此相反，生理缓冲液（含有〜100mM的盐）与一低电压，500赫兹AC场工作良好，但需要一个长期（〜12小时）肿胀周期15。此方法是基于使用ITO滑动24较早的协议，但是使用自定义腔室续癌宁两个铂丝， 如图2（见用于更简单的设计细节和建议的讨论中，即兴室）。 图3示出了凝胶辅助膨胀方法。该协议可以很好地缓冲生理食盐浓度，迅速，并且产生GUVs具有更均匀的蛋白质分布。然而，分离的，显然无缺陷GUVs收率（ 即 GUV膜是均匀的，在光学长度尺度，并且不括任何对象）较低，但它提供了足够数量的用于膜片钳和显微操作实验。此方法是基于使用琼脂糖凝胶，以产生脂质仅GUVs 16和需要更少的专门的设备比electroformation方法的协议。 GUVs用荧光显微镜的表征被描述，以及使用标准膜片钳设置到程序衡量KvAP活动“由内而外”切膜的补丁。 生长含蛋白GUVs可以比脂质仅GUVs更加困难。尤其是，最后的GUV收率上究竟如何在SUV溶液沉积和脱水以形成薄膜叠层灵敏依赖。有人未经任何以往的经验与GUVs，它可以是下面的其中隔膜是通过从有机溶剂沉积类脂形成的常规协议15,16到第一生长脂质仅GUVs很有帮助。一旦现有协议工作良好，SUV沉积和部分脱水然后可以使用脂质仅越野车，这也是非常有益的调整协议的新的脂质组合物时掌握。当GUVs脂质只SUV的可靠增长，它是那么只是一小步，产生含蛋白质GUVs从PROTEO-的SUV。

<table border="0" cellpadding="0" cellspacing="0" width="599">
	<tbody>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Name of the 
			Material/Equipment</td>
			<td style="width:132px;">Company</td>
			<td style="width:155px;">Catalog Number</td>
			<td style="width:149px;">Comments/ Description</td>
		</tr>
		<tr height="60">
			<td dir="LTR" height="60" style="height:60px;width:163px;">DPhPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine)</td>
			<td style="width:132px;">Avanti Polar Lipids&#160;</td>
			<td>850356P</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="60">
			<td dir="LTR" height="60" style="height:60px;width:163px;">Egg PC L-&#945;-phosphatidylcholine (Egg, Chicken)&#160;</td>
			<td style="width:132px;">Avanti Polar Lipids&#160;</td>
			<td>840051P</td>
			<td style="width:149px;"></td>
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		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Egg PA L-&#945;-phosphatidic acid (Egg, Chicken)</td>
			<td style="width:132px;">Avanti Polar Lipids&#160;</td>
			<td>840101P</td>
			<td style="width:149px;"></td>
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			<td dir="LTR" height="60" style="height:60px;width:163px;">18:1 (&#916;9-Cis) PC (DOPC) 1,2-dioleoyl-sn-glycero-3-phosphocholine</td>
			<td style="width:132px;">Avanti Polar Lipids&#160;</td>
			<td>850375P</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">cholesterol (ovine wool, &#62;98%)&#160;</td>
			<td style="width:132px;">Avanti Polar Lipids&#160;</td>
			<td>700000P</td>
			<td style="width:149px;"></td>
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			<td dir="LTR" height="20" style="height:20px;width:163px;">TRed-DHPE</td>
			<td style="width:132px;">Invirtogen</td>
			<td>T-1395MP</td>
			<td style="width:149px;">labeled lipid</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">BPTR-Ceramide</td>
			<td style="width:132px;">Invirtogen</td>
			<td>D-7540&#160;</td>
			<td style="width:149px;">labeled lipid</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Choloroform</td>
			<td style="width:132px;">VWR</td>
			<td>22711.290</td>
			<td style="width:149px;">AnalaR Normapur</td>
		</tr>
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			<td dir="LTR" height="20" style="height:20px;width:163px;">Acetone</td>
			<td style="width:132px;">VWR</td>
			<td>20066.296</td>
			<td style="width:149px;">AnalaR Normapur</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Ethanol</td>
			<td style="width:132px;">VWR</td>
			<td>20821.330</td>
			<td style="width:149px;">AnalaR Normapur</td>
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		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Kimwipe</td>
			<td style="width:132px;">Kimtech</td>
			<td>7552</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Hamilton syringes&#160;</td>
			<td style="width:132px;">Hamilton Bonaduz AG</td>
			<td></td>
			<td style="width:149px;">diverse</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Amber vials and teflon cups</td>
			<td style="width:132px;">Sigma</td>
			<td>SU860083 and SU860076</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Parafilm</td>
			<td style="width:132px;">VWR</td>
			<td>291-1214</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">microcentrifuge tube</td>
			<td style="width:132px;">eppendorf</td>
			<td></td>
			<td style="width:149px;">diverse</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Agarose</td>
			<td style="width:132px;">Euromex</td>
			<td style="width:155px;">LM3 (1670-B, Tg 25.7C, Tm 64C)</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Sucrose</td>
			<td style="width:132px;">Sigma</td>
			<td>84097-1kg</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Glucose&#160;</td>
			<td style="width:132px;">Sigma</td>
			<td>G8270-1kg</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Trehalose</td>
			<td style="width:132px;">Sigma</td>
			<td>T9531-25G</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">KCl</td>
			<td style="width:132px;">Sigma</td>
			<td>P9333-1kg</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Hepes</td>
			<td style="width:132px;">Sigma</td>
			<td>H3375-100G</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">TRIS</td>
			<td style="width:132px;">Euromex</td>
			<td>EU0011-A</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Osmometer</td>
			<td style="width:132px;">Wescor</td>
			<td>Vapro</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">pH meter</td>
			<td style="width:132px;">Schott instruments</td>
			<td>Lab850</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Sonicator</td>
			<td style="width:132px;">Elmasonic</td>
			<td>S 180 H</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Syringe filter 0.2&#181;m</td>
			<td style="width:132px;">Sartorius steim</td>
			<td>Minisart 16532</td>
			<td style="width:149px;"></td>
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		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Function generator</td>
			<td style="width:132px;">TTi</td>
			<td>TG315</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Platinum wires</td>
			<td style="width:132px;">Goodfellow</td>
			<td>LS413074</td>
			<td style="width:149px;">99.99+%, d=0.5mm</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Polytetrafluoroethylene</td>
			<td style="width:132px;">Goodfellow</td>
			<td></td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Dow Corning &#39;high vacuum grease&#39;</td>
			<td style="width:132px;">VWR</td>
			<td>1597418</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">sealing paste</td>
			<td style="width:132px;">Vitrex medical A/S, Denmark</td>
			<td>REF 140014</td>
			<td style="width:149px;">Sigillum Wax</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Cover slides 22x40 mm No1,5</td>
			<td style="width:132px;">VWR&#160;</td>
			<td>631-0136</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Cover slides 22x22 mm No1,5</td>
			<td style="width:132px;">VWR&#160;</td>
			<td>631-0125</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">plasma cleaner</td>
			<td style="width:132px;">Harrick</td>
			<td>PDC-32G</td>
			<td style="width:149px;">airplasma, setting &#39;high&#39;</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">petri dishes</td>
			<td style="width:132px;">Falcon BD</td>
			<td>REF 353001</td>
			<td style="width:149px;">3.5 cmx1cm</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">patch clamp amplifier</td>
			<td style="width:132px;">Axon instruments</td>
			<td>Multiclamp700B</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">DAQ Card</td>
			<td style="width:132px;">National Instruments</td>
			<td>PCI-6221</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Labview</td>
			<td style="width:132px;">National Instruments</td>
			<td>version 8.6</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="60">
			<td dir="LTR" height="60" style="height:60px;width:163px;">Glass pipettes boro silicate OD 1mm ID 0.58mm</td>
			<td style="width:132px;">Harvard Apparatus</td>
			<td>GC100-15</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Electrode holder</td>
			<td style="width:132px;">Warner Instruments&#160;</td>
			<td>Q45W-T10P</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Micromanipulator</td>
			<td style="width:132px;">Sutter&#160;</td>
			<td>MP-285</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">pipette puller</td>
			<td style="width:132px;">Sutter&#160;</td>
			<td>P-2000&#160;</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Camera</td>
			<td style="width:132px;">ProSilica&#160;</td>
			<td>GC1380</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Zeiss microscope</td>
			<td style="width:132px;">Zeiss</td>
			<td>Axiovert 135</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="60">
			<td dir="LTR" height="60" style="height:60px;width:163px;">Objective</td>
			<td style="width:132px;">Zeiss</td>
			<td></td>
			<td style="width:149px;">40x long working distance, Phase contrast</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Objective&#160;</td>
			<td style="width:132px;">Zeiss</td>
			<td></td>
			<td style="width:149px;">100x Plan-Apochromat NA 1.3</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Filterset GFP (for Alexa-488)</td>
			<td style="width:132px;">Horiba</td>
			<td>XF100-3</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Filterset Cy3 (for TexasRed)</td>
			<td style="width:132px;">Horiba</td>
			<td>XF101-2</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">beta-casein</td>
			<td style="width:132px;">Sigma&#160;</td>
			<td>C6905-1G</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">confocal microscope</td>
			<td style="width:132px;">Nikon Eclipse TE 2000-E</td>
			<td></td>
			<td style="width:149px;">D-Eclipse C1 confocal head</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Objective</td>
			<td style="width:132px;">Nikon</td>
			<td></td>
			<td style="width:149px;">Plan Fluor 100&#215; NA1.3</td>
		</tr>
		<tr height="60">
			<td dir="LTR" height="60" style="height:60px;width:163px;">matlab</td>
			<td style="width:132px;">Mathworks</td>
			<td></td>
			<td style="width:149px;">for image processing and analysis of the current traces</td>
		</tr>
	</tbody>
</table>

reconstitution of a transmembrane protein, the voltage-gated ion channel, kvap, into giant unilamellar vesicles for microscopy and patch clamp studies

巨人单层囊泡（GUVs）是一种流行的仿生系统研究膜相关现象。然而，通常使用的协议来生长GUVs必须为了形成含有官能跨膜蛋白GUVs进行修改。本文介绍了两种脱水 - 再水化方法 - electroformation和凝胶辅助肿胀 - 以形成含有所述电压 - 门控钾通道，KvAP GUVs。在两种方法中，对含有蛋白质的小单层囊泡的溶液是部分脱水，形成一个叠层膜，然后使其在再水化缓冲液溶胀。为electroformation方法，所述膜沉积在铂电极以使得AC场可以在膜补液施加。与此相反，凝胶溶胀辅助方法使用琼脂糖凝胶基质，以提高膜补液。这两种方法都可以产生在低（ 例如，5毫米）和生理（ 例如，100毫摩尔）的盐浓度GUVs。所得GUVs的特征在于通过荧光显微镜，和使用该内面向外膜片钳构造重构信道的函数进行测量。而以交替的电场（electroformation）的存在下溶胀得到高产率的无缺陷GUVs，凝胶辅助溶胀方法产生更均匀的蛋白质的分布，不需要特殊的设备。

GUVs的生长可通过检查生长室，在显微镜下进行快速评估。对于electroformation，所述GUVs倾向于增长沿铂导线束， 如图4。在凝胶辅助肿胀，GUVs显示为球形的结构，可以快速成长，并融合在一起（ 图5）。 无缺陷GUVs更容易识别和传送到一个​​观察室之后进行评价。需要校准测量来严格评价GUV质量，并且一个系统的量化先前已公布的28。然而，?...

Watch this Scientific Journal Video about Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies at JoVE

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

跨膜蛋白，KvAP，变成巨大的单层囊泡（GUVs）的重组被证明两脱水补液方法 - electroformation和凝胶辅助肿胀。在两种方法中，将含有蛋白的小单层囊泡融合在一起，以形成可以随后通过荧光显微镜和膜片钳电生理学来研究GUVs。

跨膜蛋白的重组，电压门控离子通道，KvAP，进入巨人单层囊的显微镜和膜片钳研究

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Research

JoVE Journal

Biology

Methods for Patch Clamp Capacitance Recordings from the Calyx

膜片钳从花萼电容录音方法

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. ...

科研

生物学

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

补丁钳爪蟾卵母细胞中表达的离子通道

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

优化转染的HEK - 293T细胞中的表达和重组的电压门控离子通道的电生理记录策略

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) ...

Measuring Peptide Translocation into Large Unilamellar Vesicles

测量肽转运到大Unilamellar囊泡

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1.  Many of these are referred ...

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

探讨动脉平滑肌的KV7钾离子通道功能使用膜片钳电生理和压力Myography

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

Examining BCL-2 Family Function with Large Unilamellar Vesicles

检查Bcl-2家族大单层脂质体的功能

The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate ...

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

巨脂质体制剂成像和膜片钳电

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify ...

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

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 ...