pAnap是Peter Schultz博士（斯克里普斯研究所）的礼物。这项工作由加拿大卫生研究所MOP-102689和MOP-136894（加拿大RB）和加拿大创新基金会950-225005资助。

青蛙操作按照加拿大指南进行，并经蒙特利尔大学道德委员会（CDEA，协议＃15-042）的批准。 1. fUAA公司的mRNA准备<ol><li>选择蛋白质感兴趣的位置，预期会发生构象变化。选择该区域中的氨基酸代替fUAA。 注意:位置的选择是基于预期的结构重新排列。如果存在高分辨率结构和预期运动的假设，则应该放置该化学物质的环境将会改变;这可能是介电常数（疏水性与亲水性环境）的变化，或者更可能是由另一种氨基酸猝灭。最好的猝灭剂是色氨酸。应该在一个状态（范德瓦尔半径的重叠）与猝灭剂接触，并将其与另一个。如果不存在高分辨率结构或模型，则必须扫描感兴趣的区域。在任一情况下，建议选择几个附近的位置以增加获得表达和荧光信号的概率。为了最小化蛋白质成熟和/或功能期间的空间效应，可以选择取代大和芳香族氨基酸（Phe，Trp，Tyr）。然而，作者经历了扫描fUAA插入的感兴趣区域，而不管取代的氨基酸如何，更有效率。 </li><li>在选定的位点使用定点突变15插入琥珀终止密码子（TAG）。确保感兴趣的蛋白质不以琥珀终止密码子（TAG）结束。如果是这样，突变为不同的（赭色或蛋白石终止密码子）。扩增，分离和序列DNA。通过体外转录16获得蛋白质mRNA，并将mRNA存储在20℃或80℃。 </li> &lt;/醇&gt; 2.卵母细胞的制备和注射<ol><li> 手术从非洲爪蟾青蛙手术获得V或VI卵母细胞，如前所述17胶原酶脱胶。 <ol><li>根据批准的动物方案（本文为:3-氨基苯甲酸乙酯），用合适的麻醉剂麻醉青蛙。当他们不能轻蔑地对脚尖做出反应（失去撤回反射）时，则将其适当地麻醉以进行手术。 </li><li>立即从麻醉溶液中取出青蛙，用清水彻底冲洗皮肤。这种冲洗将通过从皮肤表面去除未吸收的化学物质来防止动物进入更深层次的麻醉。 </li><li>从一侧手术切除卵巢节点，并使用两个镊子仔细打开节点。孵育并搅拌含有1％（w / v）胶原酶的"标准卵母细胞溶液"（SOS）中的卵母细胞20-30分钟去流产用SOS溶液洗三次。 </li><li>分别选择大型和健康的卵母细胞，并在注射前至少4小时将其添加到补充有抗生素（100U / mL青霉素，100μg/ mL链霉素，10mg / 100mL卡那霉素）和5％马血清的Barth溶液中至少4小时。 注意:经2-4次延迟的2 - 4次手术之后，通过与3-氨基苯甲酸乙酯长时间（&gt; 1小时）孵育非洲爪蟾进行安乐死。 </li></ol></li><li> 对于DNA的核注射，准备长而薄的注射针头以能够到达细胞核并避免损伤卵母细胞。用注射嘴填充油并将其安装在纳米注射器上。 <ol><li>在立体显微镜下安装纳米注射器，并使用镊子打破尖端的末端。排出油，直到没有气泡陷入尖端的末端。 </li></ol></li><li>将1μL0.1μg/＃181;将L pAnap在含有NaOH（1％的1N NaOH）的无核酸酶的水上，在立体镜下在一片石蜡膜上，并用DNA填充注射尖端。 </li><li>将40个卵母细胞转移到含有补充有抗生素的Barth溶液的网状包被的注射皿中。 注意:为了制作网眼注射皿，切出适当尺寸的800μm尼龙网填充聚苯乙烯培养皿。将氯仿加入中心，然后将网格放在顶部。将网格平直，直到塑胶套。 </li><li>由于卵母细胞核位于动物（黑暗）极点，将注射针瞄准动物杆的中心并使其尖端到达动物半球的中心附近（或与RNA注射相比2-3倍的深度） ）。将每个卵母细胞中的pAnap注入9.2 nL。薄的尖端和小的注射体积可能导致不规则的注射或阻塞的尖端。偶尔检查注射是否注入空气。 注意:DNA是否适当地注入细胞核是不确定的。因此，期望10-40％的卵母细胞不表达tRNA /合成酶对。参见讨论进一步阐述。 </li></ol><img alt="图2" src="/files/ftp_upload/55598/55598fig2.jpg" /> 图 2 :DNA和RNA注射入 非洲爪蟾 卵母细胞的插图。 首先，将pAnap注射入非洲爪蟾卵母细胞核（ 1 ）。 6-24h后，将Anap和通道RNA共同植入植物极（ 2 ）。通过由pAnap编码的氨酰-tRNA合成酶，Anap将与携带琥珀终止反密码子的tRNA正交氨基酰化。这样，氨基酰化的Anap-tRNA被通道中插入的琥珀终止密码子的核糖体识别1 RNA，导致终止密码子的抑制和Anap的插入。 <a href="https://www.jove.com/files/ftp_upload/55598/55598fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <ol start="6"><li>在2℃的Barth溶液中孵育卵母细胞，补充抗生素和5％马血清（HS）在18℃下孵育6-24小时，以使得能够稳定表达Anap特异性tRNA和tRNA合成酶。 注意:DNA孵育时间可以在注射RNA前几天持续，但不会增加表达。 </li><li>准备用于RNA注射的纳米注射器（与步骤2.2相同，但注射头不需要像DNA注射那样薄）。此时只能在红灯下工作，以防止Anap的漂白。 </li><li>将1μL1 mM Anap与1μL1-2μg/μLmRNA直接混合在一块石蜡膜上，并用混合溶液填充注射头。在植物的膜下方的Im​​pale（亮）极，并在每个注射apAap注射的卵母细胞中注射46nL。 注意:所需的mRNA浓度取决于感兴趣的蛋白质。 </li><li>孵育保存在盒子中的卵母细胞或包裹在铝箔中，在Barth溶液中补充抗生素和5％马血清，18℃下2-3天。每天与新鲜的Barth溶液进行交换，并去除死卵卵母细胞以避免污染。 </li></ol> 3. VCF设置<ol><li>如前所述安装切开的卵母细胞电压钳设备18 。 </li><li>将电生理学记录系统安装在立式荧光显微镜上，将记录室安装在滑块上，允许在标准立体镜之间移动它以放置卵母细胞和显微镜进行荧光测量（ 图2c ）。 注意:切开的卵母细胞的几何形状不适合使用正常的tra在操作过程中记录光照明。因此，使用带有红色滤光片的"鹅颈式"卤素灯从顶部向侧面照亮。可以去除显微镜的冷凝器，使空间降低电生理室的阶段。 </li><li>将光电二极管检测系统连接到荧光显微镜的C-mount出口端口（ 图2a ）。将光电流读数连接到数字信号处理器（DSP，模拟/数字 - 数字/模拟转换器）中的第二个输入通道。 </li><li>使用100 W，12 V卤素灯作为荧光激发的光源。 注意:或者，可以使用Hg-燃烧器，但必须降低强度，以防止在记录期间太快的光漂白。只有当各个LED在激发范围内显示出显着的强度（ 例如， Anap的〜350 nm）时才推荐使用LED照明。大多数白光LED不能进入紫外光谱。 </li><li>插件在激励光源和显微镜之间设置电控快门，并将其控制（通常为TTL脉冲）连接到DSP的数字输出。在记录软件中记录TTL脉冲（参见制造商说明书），使快门在录制开始之前〜100 ms开始。这样，打开过程中的任何振动都不会影响记录。时间取决于快门的速度和振动。在记录结束前结束脉冲5ms， 如图4所示 。这样，也记录了总荧光的值。 </li><li>在过滤器立方体塔架中插入适当的过滤器立方体（激发过滤器，分色镜和发射过滤器）。对于Anap，使用Ex:377/50 nm带通，二色409 nm长通和Em:470/40 nm带通。 </li></ol><img alt="图3" src="/files/ftp_upload/55598/55598fig3.jpg" /> 图3<html> / strong&gt; :VCF设置。 （ A ）VCF设置的侧视图，显示显微镜内的光路。过滤器立方体包含激发滤光片，分色镜和发射滤光片。 （ B ）选择的卵母细胞室尺寸为上室半径（1）为3.4厘米，底室长度为2厘米为5.5厘米，底室宽度为3厘米，中间室宽度为4厘米为4厘米。 （ C ）VCF设置的前视图。左侧的第一个眼睛是将卵母细胞安装在切开的电压钳夹室中并用于透化。然后，在右侧的第二次眼镜下，在显微镜下滑动腔室。这里，使用4X物镜将V1电极插入卵母细胞，并使用浸水式40X物镜记录荧光。 <a href="https://www.jove.com/files/ftp_upload/55598/55598fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> itle"&gt; 4。VCF录音<ol><li>按照前面所述的可视化开放式卵母细胞电压钳的准备步骤18 （琼脂桥制备，安装卵母细胞，皂角苷透化）。然而，在红灯下始终工作，以避免在记录之前漂白荧光团。放置卵母细胞时，请确保动物杆朝上。动物极膜下的色素沉着屏蔽来自细胞溶质的自发荧光，因此降低背景荧光。 </li><li>将腔室滑动到显微镜并使用4X物镜进行对焦。 </li><li>用电压感应V1电极（3 M KCl）冲击卵母细胞，切换到40X浸水（NA 0.8 - 0.9）的目标。专注于面向上的动物杆。 </li><li>关掉红灯。通过转动过滤器立方体塔架和连接到光电二极管的光学出口端口，选择正确的过滤器立方体。打开halogen灯处于最高强度，并且快速打开快门2-5秒以读取源自卵母细胞的背景荧光强度。通过描述的设置，Anap的值应该在50-200 pA左右。 </li><li>打开夹子，将浴室/护罩开关翻转到活动状态，并转动I台灯上的旋钮将膜电位（V1 - V2）调整到指令电位。 </li><li>在录音软件中选择保持电位，步进协议，脉冲数量和长度等。记录电压依赖电流和Anap荧光强度。 </li></ol>双色VCF <ol><li>要同时监测同一蛋白中的两个位置，将细胞外和可及的氨基酸突变为半胱氨酸，并除去其他半胱氨酸，以确保用硫醇化学特异性标记。 </li><li>执行步骤2.1-2.5。 </li><li>在VCF记录之前，将5μMTMR-马来酰亚胺的卵母细胞在标记溶液中孵育15分钟（或其他染料与Anap相比具有非重叠光谱）。 </li><li>用标记溶液洗涤卵母细胞三次以除去多余的染料。 </li><li>执行步骤4.1-4.6。 </li><li>在过滤器立方体塔架中插入适当的TMR滤波器（激发滤光片，分色镜和发射滤光片）。转动过滤塔，切换到TMR过滤器立方体。 </li><li>读取TMR的背景荧光，如步骤4.4中Anap所述。 注意:由于膜上的非特异性标记，硫醇化学的标记导致高背景荧光。因此，TMR背景荧光可能使放大器饱和（&gt; 2,000 pA）。在这种情况下，不要降低光强度，而是通过向光电二极管添加偏移电流来简单地减去背景荧光。在市售系统中，使用检测器系统上的"采样保持"功能。注意实验室j中的背景荧光值（使用10X中性密度滤光片）ournal，因为这个值不会被记录（饱和）。 </li><li>按照步骤4.6同时记录电压相关电流和TMR荧光强度。 </li></ol>

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(85), (2014).</li><li>Zhao, J., Blunck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolated+voltage+sensing+domain+of+the+Shaker+potassium+channel+forms+a+voltage-gated+cation+channel.">The isolated voltage sensing domain of the Shaker potassium channel forms a voltage-gated cation channel.</a> Elife. 5, (2016).</li><li>Posson, D. J., Ge, P., Miller, C., Bezanilla, F., Selvin, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+vertical+movement+of+a+K++channel+voltage+sensor+measured+with+luminescence+energy+transfer.">Small vertical movement of a K+ channel voltage sensor measured with luminescence energy transfer.</a> Nature. 436 (7052), 848-851 (2005).</li><li>Chanda, B., Asamoah, O. K., Blunck, R., Roux, B., Bezanilla, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gating+charge+displacement+in+voltage-gated+ion+channels+involves+limited+transmembrane+movement.">Gating charge displacement in voltage-gated ion channels involves limited transmembrane movement.</a> Nature. 436 (7052), 852-856 (2005).</li><li>Taraska, J. W., Puljung, M. C., Zagotta, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-distance+probes+for+protein+backbone+structure+based+on+energy+transfer+between+bimane+and+transition+metal+ions.">Short-distance probes for protein backbone structure based on energy transfer between bimane and transition metal ions.</a> Proc Natl Acad Sci U S A. 106 (38), 16227-16232 (2009).</li><li>Baker, B. J., 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=Genetically+encoded+fluorescent+sensors+of+membrane+potential.">Genetically encoded fluorescent sensors of membrane potential.</a> Brain Cell Biol. 36 (1-4), 53-67 (2008).</li><li>Miranda, 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=State-dependent+FRET+reports+calcium-+and+voltage-dependent+gating-ring+motions+in+BK+channels.">State-dependent FRET reports calcium- and voltage-dependent gating-ring motions in BK channels.</a> Proc Natl Acad Sci U S A. , (2013).</li><li>Sisido, M., Ninomiya, K., Ohtsuki, T., Hohsaka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four-base+codon/anticodon+strategy+and+non-enzymatic+aminoacylation+for+protein+engineering+with+non-natural+amino+acids.">Four-base codon/anticodon strategy and non-enzymatic aminoacylation for protein engineering with non-natural amino acids.</a> Methods. 36 (3), 270-278 (2005).</li><li>Hohsaka, T., Ashizuka, Y., Murakami, H., Sisido, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Five-base+codons+for+incorporation+of+nonnatural+amino+acids+into+proteins.">Five-base codons for incorporation of nonnatural amino acids into proteins.</a> Nucleic Acids Res. 29 (17), 3646-3651 (2001).</li></ol>

作者没有什么可以披露的。

与tRNA合成酶连续转录的tRNA 的体内氨基酰化使得可以获得荧光测量的高表达水平。为了有效的fUAA并入，pAnap正确注入细胞核是至关重要的。由于核的确切位置的不确定性，预计10-40％的DNA注射会失败，导致不表达（或泄漏表达）卵母细胞。因此，在没有Anap和pAnap的情况下检查表达是重要的，以识别泄漏通道情况下的当前表型和幅度。这样，通过适当的DNA注射（高表达，Anap dF，全长当前表型?...

具有各种化学和物理性质的200多种非天然氨基酸被遗传并入大肠杆菌 ，酵母和哺乳动物细胞中的蛋白质3 。非天然氨基酸通过正交工程化的tRNA /合成酶对而与特异性终止密码子相结合。改变蛋白质的遗传学方法为蛋白质结构和功能提供了宝贵的见解。在这里，我们提出使用电压钳夹荧光测量（VCF）与荧光UAA组合的方案。 在VCF中，同时观察位于荧光探针周围的功能数据和结构重排（〜5Å）可使我们以毫秒级分辨率获得动态信息1 。荧光探针在蛋白质定位运动时改变其淬灭状态。只有1-2埃的运动足以导致荧光的显着变化强度4 。鉴定目标蛋白质感兴趣位点后，通过点突变突变位点。经典地，残基已被突变为半胱氨酸，而现在，引入琥珀终止密码子（TAG）用于遗传fUAA并入。然后蛋白质被体外转录。 虽然其他表达系统（ 例如哺乳动物细胞）可以使用5,6,7 ， 非洲爪蟾卵母细胞因结构功能研究而较大，因为其尺寸较大，导致操作更容易，荧光强度更高（更多的荧光团），因此，信噪比。此外， 非洲爪蟾卵母细胞具有低内源性蛋白质的背景2,8，动物杆上的黑色色素沉着抵抗来自t的背景荧光他的细胞质手术切除非洲爪蟾卵母细胞，将编码fUAA特异性的正交tRNA / tRNA-合成酶对的DNA注射到卵母细胞核中。在孵育6-24小时后，将蛋白质RNA与fUAA共注射到卵母细胞的细胞溶质中，随后孵育2-3天。为了防止对fUAA（光漂白）的任何损坏，包括Anap在内的程序必须在红光下进行，以避免荧光团激发。 在切开的卵母细胞电压钳设置上研究卵母细胞，其安装在直立荧光显微镜上，电流和荧光变化同时记录9,10 。或者，可以使用双电极电压钳1或膜片钳构造11 。荧光被具有低RMS噪声的适当波长激发<html>使用连接到具有高放大率的放大器的光电二极管记录的发射。 在电压钳荧光测定中使用荧光非天然氨基酸（fUAAs）有几个优点。一个是进入膜蛋白质的胞质侧;许多调节过程位于这里（ 例如， Ca 2+ - 或核苷酸结合位点，电压门控离子通道的快速和闭态失活，开孔，模块耦合）。所有这些过程现在都可用于荧光标记。 另一个优点是探针的小尺寸导致蛋白质的较少干扰。到目前为止，fUAAs的两个正交tRNA / tRNA合成酶对已被设计为12,13 ，其中3-（6-乙酰基萘-2-基氨基）-2-氨基丙酸（Anap）是唯一已被用于非洲爪蟾卵母细胞的fUAA 如图2所示 ，"xref"&gt; 8。 Anap是一种环境敏感的荧光团，分子量为272.3 g / mol，仅略高于色氨酸12 （ 图1A，1B ）。由于其小的尺寸，与通过接头连接的常规荧光团（通常大于500g / mol）相比，荧光团可能引入较少的空间效应。此外，在Anap的情况下，荧光团比与半胱氨酸相连的位置更靠近蛋白质主链，因此，Anap正在探测更局部的重排。最后，在UAA-VCF中不再需要在常规VCF中去除内源性半胱氨酸以确保位点特异性标记，因此（i）使蛋白质（几乎）为其天然状态，（ii）允许VCF应用研究更广泛的蛋白质，其功能可能被半胱氨酸取代改变。 <img alt="图1" src ="/ files / ftp_upload / 55598 / 55598fig1.jpg"/&gt; 图1 :Anap和荧光光谱。 （ A ）Anap的化学结构。 （ B ）1nM Anap的归一化吸收光谱和发射光谱，证明Anap荧光对溶剂疏水性的敏感性。通过在350nm激发获得发射光谱。 <a href="https://www.jove.com/files/ftp_upload/55598/55598fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 使用荧光UAA的缺点是如果氨基酰化tRNA的量稀少，则异源蛋白质群体可能由终止密码子读取，翻译重新启动，C-末端截短的蛋白质或内源氨基酰化的串扰引起。在不存在fUAA和tRNA / tRNA合成酶对的情况下，应始终检查这种泄漏表达。我们处理了转交的问题国际重新启动以及如何规避N末端插入位置以前14 。然而，当fUAA，tRNA和tRNA合成酶以饱和量存在时，仅存在泄漏表达的可能性较低。 fUAA-VCF与常规VCF的关键程序差异在于注射和处理卵母细胞;注射编码tRNA和tRNA合成酶（pAnap）的DNA之后，引入Anap，其与蛋白质mRNA共注射，或者作为乙酰氧基甲基（AM）酯加入到孵育溶液中。

<table border="0" cellpadding="0" cellspacing="0" width="567">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">Solutions</td>
			<td style="width:111px;"></td>
			<td style="width:161px;"></td>
			<td style="width:96px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Barth&#39;s solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td>90mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td>Fisher Scientific</td>
			<td>BP366-500</td>
			<td>3mM</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">MgSO4</td>
			<td>Sigma-Aldrich</td>
			<td>M-9397</td>
			<td>0.82mM</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C-7902</td>
			<td>0.41mM</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Ca(NO3)2</td>
			<td>Sigma-Aldrich</td>
			<td>C-1396</td>
			<td>0.33mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td>5mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaOH hydrate</td>
			<td>BDH</td>
			<td>BDH7225-4</td>
			<td>pH 7.6</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicilin</td>
			<td>Invitrogen</td>
			<td>15140122</td>
			<td>100U/mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140122</td>
			<td>100&#181;g/mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kanamycin</td>
			<td>Invitrogen</td>
			<td>15160054</td>
			<td>10mg/100mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Horse serum</td>
			<td>Invitrogen</td>
			<td>16050122</td>
			<td>5%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SOS Standard Oocyte Solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>746398</td>
			<td>102 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td>Sigma-Aldrich</td>
			<td>746436</td>
			<td>3 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M9272</td>
			<td>1 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td>5 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">External recording solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N-methyl-D-glucamine (NMDG)</td>
			<td>Alfa Aesar</td>
			<td>L14282</td>
			<td>115mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td>10mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>239232</td>
			<td>2mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MES hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>258105</td>
			<td>pH 7.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Internal recording solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N-methyl-D-glucamine (NMDG)</td>
			<td>Alfa Aesar</td>
			<td>L14282</td>
			<td>115mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td>10mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethylenediamine Tetraacetic Acid (EDTA)</td>
			<td>Fisher Scientific</td>
			<td>E478-500</td>
			<td>2mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MES hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>258105</td>
			<td>pH 7.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Labeling solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KOH</td>
			<td>Fisher Scientific</td>
			<td>P250-1</td>
			<td>115mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td>10mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>239232</td>
			<td>2mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MES hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>258105</td>
			<td>pH 7.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TMR stock solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tetramethylrhodamine-5-maleimide (TMR)</td>
			<td>Molcular Probes by Life Technologies</td>
			<td>T6027</td>
			<td>5mM in DMSO</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anap stock solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;">Anap</td>
			<td>ABZENA (TCRS)</td>
			<td>Custom synthesis TCRS-170</td>
			<td style="width:96px;">1mM in nuclease-free water and 1% NaOH 1N</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Material/Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pAnap</td>
			<td>Addgene</td>
			<td>48696</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High Performance Oocyte Clamp</td>
			<td>Dagan Corporation</td>
			<td>CA-1B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gpatch Acquisition software</td>
			<td colspan="3">Department of Anesthesiology, University of California, Los Angeles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Analysis software</td>
			<td colspan="3">Department of Anesthesiology, University of California, Los Angeles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Recording Chamber</td>
			<td colspan="2">Custom machined</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Photo diode detection system</td>
			<td>Dagan Corporation</td>
			<td>PhotoMax-200/PIN</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electrical shutter driver</td>
			<td>UNIBLITZ</td>
			<td>VCM-D1</td>
			<td></td>
		</tr>
	</tbody>
</table>

voltage-clamp fluorometry in xenopus oocytes using fluorescent unnatural amino acids

电压钳式荧光法（VCF）一直是研究电生物膜蛋白的结构和功能的选择技术，其中荧光和电流的实时测量同时报告局部重排和全局功能。虽然高分辨率结构技术如低温电子显微镜或X射线晶体学提供了感兴趣的蛋白质的静态图像，但VCF提供了动态结构数据，使我们能够将结构重排（荧光）与动态功能数据（电生理学）联系起来。直到最近，用于蛋白质定点荧光标记的硫醇反应性化学物质限制了该方法的范围，因为所有可及的半胱氨酸，包括内源性半胱氨酸都将被标记。因此需要构建不含内源性半胱氨酸的蛋白质。标记也限于可从细胞外获得的位点侧。随着使用荧光非天然氨基酸 （fUAA），使用正交tRNA和tRNA合成酶对2特异性并入小荧光探针以响应终止密码子抑制而改变。 VCF改进仅需要DNA注射（tRNA /合成酶对）的两步注射程序，然后进行RNA / fUAA共注射。现在，标记细胞内和掩埋的位点是可能的，VCF的使用已经显着扩大。因此，VCF技术对于研究广泛的蛋白质变得有吸引力，更重要的是允许研究许多细胞溶质调节机制。

图4显示了在存在pAnap和Anap的情况下，从表达快速灭活快速灭活的卵母细胞（IR），L382stop-W434F的卵母细胞获得的VCF记录的实例。 W434F突变阻断离子钾电流，这使得可以测量瞬态门控电荷位移（门电流）。门极电流（上限曲线）和Anap荧光强度变化（下限轨迹）同时记录去极化表明Anap成功纳入了位置L382。在这里，Anap位于在激活期间移动的四个S4跨膜螺旋中的每?...

Watch this Scientific Journal Video about Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids at JoVE.com

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

本文介绍了使用荧光非天然氨基酸（fUAA）代替马来酰亚胺染料的常规电压钳子荧光测量（VCF）的增强，以探测离子通道中的结构重排。该程序包括非洲爪蟾卵母细胞DNA注射，RNA / fUAA共注射，以及同步电流和荧光测量。

电压钳荧光测定<em&gt;爪蟾</em&gt;使用荧光非天然氨基酸的卵母细胞

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where ...

voltage-clamp-fluorometry-xenopus-oocytes-using-fluorescent-unnatural

Research

JoVE Journal

Biochemistry

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

10.4K 次观看.  University of Montréal. 本文介绍了使用荧光非天然氨基酸（fUAA）代替马来酰亚胺染料的常规电压钳子荧光测量（VCF）的增强，以探测离子通道中的结构重排。该程序包括非洲爪蟾卵母细胞DNA注射，RNA / fUAA共注射，以及同步电流和荧光测量。 

视频: Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

A Polyaniline-based Sensor of Nucleic Acids

Detection of nucleic acids is at the center of diagnostic technologies used in research and the clinic. Standard approaches used in these technologies rely on enzymatic modification that can introduce bias and artifacts. A critical element of next generation detection platforms will be direct molecular sensing, thereby avoiding a need for amplification or labels. Advanced nanomaterials may provide the suitable chemical modalities to realize label-free sensors. Conjugated polymers are ideal for biological sensing, possessing properties compatible with biomolecules and exhibit high sensitivity to localized environmental changes. In this article, a method is presented for detecting nucleic acids using the electroconductive polymer polyaniline. Simple DNA "probe" oligonucleotides complementary to target nucleic acids are attached electrostatically to the polymer, creating a sensor system that can differentiate single nucleotide differences in target molecules. Outside the specific and unbiased nature of this technology, it is highly cost effective.

Nucleic acids are common analytes for assessing biological systems; however, bias from enzymatic manipulation can cause concern. Here a method is described for label-free detection of nucleic acids using polyaniline. This sensitive, cost-effective sensor technology can distinguish single nucleotide differences between molecules.

核酸的聚苯胺传感器

Detection of nucleic acids is at the center of diagnostic technologies used in research and the clinic. Standard approaches used in these technologies ...

科研

生物化学

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2G73E protein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2G73E proteins) as a biological example.

We describe here an optimized protocol of fluorescent Electrophoretic Mobility Shift Assays (fEMSA) using purified SOX-2 proteins together with infrared fluorescent dye-labeled DNA probes as a case study to tackle an important biological question.

一种优化的协议电泳迁移分析使用红外荧光染料标记的寡核苷酸

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. ...

Identification of Fatty Acids in Bacillus cereus

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the double bond. Changes in FA composition play a crucial role in the adaptation of bacteria to their environment. These modifications entail a change in the ratio of iso versus anteiso branched FAs, and in the proportion of unsaturated FAs relative to saturated FAs, with double bonds created at specific positions. Precise identification of the FA profile is necessary to understand the adaptation mechanisms of Bacillus species.
Many of the FAs from Bacillus are not commercially available. The strategy proposed herein identifies FAs by combining information on the retention time (by calculation of the equivalent chain length (ECL)) with the mass spectra of three types of FA derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl ester (picolinyl). This method can identify the FAs without the need to purify the unknown FAs.
Comparing chromatographic profiles of FAME prepared from Bacillus cereus with a commercial mixture of standards allows for the identification of straight-chain saturated FAs, the calculation of the ECL, and hypotheses on the identity of the other FAs. FAMEs of branched saturated FAs, iso or anteiso, display a constant negative shift in the ECL, compared to linear saturated FAs with the same number of carbons. FAMEs of unsaturated FAs can be detected by the mass of their molecular ions, and result in a positive shift in the ECL compared to the corresponding saturated FAs.
The branching position of FAs and the double bond position of unsaturated FAs can be identified by the electron ionization mass spectra of picolinyl and DMOX derivatives, respectively. This approach identifies all the unknown saturated branched FAs, unsaturated straight-chain FAs and unsaturated branched FAs from the B. cereus extract.

Identification of Fatty Acids in Bacillus cereus

We propose a protocol to identify fatty acids without the need to purify them. It combines information on the retention times with the mass spectra of three types of fatty acid derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl esters (picolinyl).

脂肪酸鉴定<em&gt;蜡状芽孢杆菌</em

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the ...

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts prepared from Xenopus laevis eggs have been powerful in dissecting biochemical networks underlying the cell-cycle progression, their ensemble average measurement typically leads to a damped oscillation, despite each individual oscillator being sustained. This is due to the difficulty of perfect synchronization among individual oscillators in noisy biological systems. To retrieve the single-cell dynamics of the oscillator, we developed a droplet-based artificial cell system that can reconstitute mitotic cycles in cell-like compartments encapsulating cycling cytoplasmic extracts of Xenopus laevis eggs. These simple cytoplasmic-only cells exhibit sustained oscillations for over 30 cycles. To build more complicated cells with nuclei, we added demembranated sperm chromatin to trigger nuclei self-assembly in the system. We observed a periodic progression of chromosome condensation/decondensation and nuclei envelop breakdown/reformation, like in real cells. This indicates that the mitotic oscillator functions faithfully to drive multiple downstream mitotic events. We simultaneously tracked the dynamics of the mitotic oscillator and downstream processes in individual droplets using multi-channel time-lapse fluorescence microscopy. The artificial cell-cycle system provides a high-throughput framework for quantitative manipulation and analysis of mitotic oscillations with single-cell resolution, which likely provides important insights into the regulatory machinery and functions of the clock.

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

We present a method for the generation of in vitro self-sustained mitotic oscillations at the single-cell level by encapsulating egg extracts of Xenopus laevis in water-in-oil microemulsions.

无细胞爪卵萃取物微乳液细胞周期振荡的重建

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts ...

Structure Solution of the Fluorescent Protein Cerulean Using MeshAndCollect

X-ray crystallography is the major technique used to obtain high resolution information concerning the 3-dimensional structures of biological macromolecules. Until recently, a major requirement has been the availability of relatively large, well diffracting crystals, which are often challenging to obtain. However, the advent of serial crystallography and a renaissance in multi-crystal data collection methods has meant that the availability of large crystals need no longer be a limiting factor. Here, we illustrate the use of the automated MeshAndCollect protocol, which first identifies the positions of many small crystals mounted on the same sample holder and then directs the collection from the crystals of a series of partial diffraction data sets for subsequent merging and use in structure determination. MeshAndCollect can be applied to any type of micro-crystals, even if weakly diffracting. As an example, we present here the use of the technique to solve the crystal structure of the Cyan Fluorescent Protein (CFP) Cerulean.

We present the use of the MeshAndCollect protocol to obtain a complete diffraction data set, for use in subsequent structure determination, composed of partial diffraction data sets collected from many small crystals of the fluorescent protein Cerulean.

利用网格和收集荧光蛋白陶瓷的结构解

X-ray crystallography is the major technique used to obtain high resolution information concerning the 3-dimensional structures of biological ...

Fluorescent Silver Staining of Proteins in Polyacrylamide Gels

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The classic silver stains have certain drawbacks, such as high background staining, poor protein recovery, low reproducibility, a narrow linear dynamic range for quantification, and limited compatibility with mass spectrometry (MS). Now, with the use of a fluorogenic Ag+ probe, TPE-4TA, we developed a fluorescent silver staining method for the total protein visualization in polyacrylamide gels. This new stain avoids the troublesome silver reduction step in traditional silver stains. Moreover, the fluorescent silver stain demonstrates good reproducibility, sensitivity, and linear quantification in protein detection, making it a useful and practical protein gel stain.

Here, we describe a detailed protocol outlining a new fluorescent staining technique for total protein detection in polyacrylamide gels. The protocol utilizes a silver ion-specific fluorescence turn-on probe, which detects Ag+-protein complexes, and eliminates certain limitations of traditional chromogenic silver stains.

聚丙烯酰胺凝胶中蛋白质的荧光银染色

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide ...

Identifying Amino Acid Overproducers Using Rare-Codon-Rich Markers

To satisfy the ever-growing market for amino acids, high-performance production strains are needed. The amino acid overproducers are conventionally identified by harnessing the competitions between amino acids and their analogs. However, this analog-based method is of low accuracy, and proper analogs for specific amino acids are limited. Here, we present an alternative strategy that enables an accurate, sensitive, and high-throughput screening of amino acid overproducers using rare-codon-rich markers. This strategy is inspired by the phenomenon of codon usage bias in protein translation, for which codons are categorized into common or rare ones based on their frequencies of occurrence in the coding DNA. The translation of rare codons depends on their corresponding rare transfer RNAs (tRNAs), which cannot be fully charged by the cognate amino acids under starvation. Theoretically, the rare tRNAs can be charged if there is a surplus of the amino acids after charging the synonymous common isoacceptors. Therefore, retarded translations caused by rare codons could be restored by feeding or intracellular overproductions of the corresponding amino acids. Under this assumption, a selection or screening system for identifying amino acid overproducers is established by replacing the common codons of the targeted amino acids with their synonymous rare alternatives in the antibiotic resistance genes or the genes encoding fluorescent or chromogenic proteins. We show that the protein expressions can be greatly hindered by the incorporation of rare codons and that the levels of proteins correlate positively with the amino acid concentrations. Using this system, overproducers of multiple amino acids can be readily screened out from mutation libraries. This rare-codon-based strategy only requires a single modified gene, and the host is less likely to escape the selection than in other methods. It offers an alternative approach for obtaining amino acid overproducers.

This study presents an alternative strategy to the conventional toxic analog-based method in identifying amino acid overproducers by using rare-codon-rich markers to achieve accuracy, sensitivity, and high-throughput simultaneously.

使用稀有的科顿富标记识别氨基酸过度生产者

To satisfy the ever-growing market for amino acids, high-performance production strains are needed. The amino acid overproducers are conventionally ...

Localization of SUMO-modified Proteins Using Fluorescent Sumo-trapping Proteins

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. This method utilizes a recombinant modified SUMO-trapping protein fragment, kmUTAG, derived from the Ulp1 SUMO protease of the stress-tolerant budding yeast Kluyveromyces marxianus. We have adapted the properties of the kmUTAG for the purpose of studying sumoylation in a variety of model systems without the use of antibodies. For the study of SUMO, KmUTAG has several advantages when compared to antibody-based approaches. This stress-tolerant SUMO-trapping reagent is produced recombinantly, it recognizes native SUMO isoforms from many species, and unlike commercially available antibodies it shows reduced affinity for free, unconjugated SUMO. Representative results shown here include the localization of SUMO conjugates in mammalian tissue culture cells and nematode oocytes.

SUMO is an essential and highly conserved, small ubiquitin-like modifier protein. In this protocol we are describing the use of a stress-tolerant recombinant SUMO-trapping protein (kmUTAG) to visualize native, untagged SUMO conjugates and their localization in a variety of cell types.

荧光苏门答腊蛋白对 sumo 修饰蛋白的定位

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. Nucleases display a variety of vital physiological roles in prokaryotic and eukaryotic organisms, ranging from maintaining genome stability to providing protection against pathogens. Altered nuclease activity has been associated with several pathological conditions including bacterial infections and cancer. To this end, nuclease activity has shown great potential to be exploited as a specific biomarker. However, a robust and reproducible screening method based on this activity remains highly desirable.
Herein, we introduce a method that enables screening for nuclease activity using nucleic acid probes as substrates, with the scope of differentiating between pathological and healthy conditions. This method offers the possibility of designing new probe libraries, with increasing specificity, in an iterative manner. Thus, multiple rounds of screening are necessary to refine the probes&#39; design with enhanced features, taking advantage of the availability of chemically modified nucleic acids. The considerable potential of the proposed technology lies in its flexibility, high reproducibility, and versatility for the screening of nuclease activity associated with disease conditions. It is expected that this technology will allow the development of promising diagnostic tools with a great potential in the clinic.

Altered nuclease activity has been associated with different human conditions, underlying its potential as a biomarker. The modular and easy to implement screening methodology presented in this paper allows the selection of specific nucleic acid probes for harnessing nuclease activity as a biomarker of disease.

使用核酸探针进行核酸活性的动力学筛选

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of methods. Here, we describe two important approaches used for this purpose. First, we describe how to enrich tissues and cells with cholesterol using cyclodextrin saturated with cholesterol using cerebral arteries (tissues) and hippocampal neurons (cells) as examples. This approach can be used for any type of tissue, cells, or cell lines. An alternative approach for cholesterol enrichment involves the use of low-density lipoprotein (LDL). The advantage of this approach is that it uses part of the natural cholesterol homeostasis machinery of the cell. However, whereas the cyclodextrin approach can be applied to enrich any cell type of interest with cholesterol, the LDL approach is limited to cells that express LDL receptors (e.g., liver cells, bone marrow-derived cells such as blood leukocytes and tissue macrophages), and the level of enrichment depends on the concentration and the mobility of the LDL receptor. Furthermore, LDL particles include other lipids, so cholesterol delivery is nonspecific. Second, we describe how to enrich Xenopus oocytes with cholesterol using a phospholipid-based dispersion (i.e., liposomes) that includes cholesterol. Xenopus oocytes constitute a popular heterologous expression system used for studying cell and protein function. For both the cyclodextrin-based cholesterol enrichment approach of mammalian tissue (cerebral arteries) and for the phospholipid-based cholesterol enrichment approach of Xenopus oocytes, we demonstrate that cholesterol levels reach a maximum following 5 min of incubation. This level of cholesterol remains constant during extended periods of incubation (e.g., 60 min). Together, these data provide the basis for optimized temporal conditions for cholesterol enrichment of tissues, cells, and Xenopus oocytes for functional studies aimed at interrogating the impact of cholesterol enrichment.

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Two methods of cholesterol enrichment are presented: the application of cyclodextrin saturated with cholesterol to enrich mammalian tissues and cells, and the use of cholesterol-enriched phospholipid-based dispersions (liposomes) to enrich Xenopus oocytes. These methods are instrumental for determining the impact of elevated cholesterol levels in molecular, cellular, and organ function.

哺乳动物组织和异种卵母细胞与胆固醇的丰富

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of ...

电压钳荧光测定

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