Name: using an &#945;-bungarotoxin binding site tag to study gaba a receptor membrane localization and trafficking
Uploaded: 2014-03-28T15:45:00
Description: Watch this Scientific Journal Video about Using an ÃŽÂ±-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking at JoVE.com

支持从匹兹堡大学医学院的药理学及化学生物学系提供启动基金。确认雅各布实验室成员谁促成了视频提交：尼古拉斯·格拉夫。

下面描述的所有协议都是按照IACUC和匹兹堡大学医学院的内部评级机构审查委员会。 1。海马神经细胞在组织培养罩的准备注意：使用无菌技术和试剂整个协议1。 <ol><li>制备聚-D-赖氨酸（0.1 mg / ml的H 2 O）涂覆的玻璃盖玻片上作为底物的神经元的培养。 <ol><li>将每个3.5厘米组织培养皿内4-5圆形玻璃盖玻片。 </li><li>点70微升聚-D-赖氨酸到每个12毫米盖玻片。注：对?...

<ol>
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此处所描述的信息为基础的固定和活的技术可以被用来跟踪受体或其它细胞质膜蛋白质运输中的细胞系，神经元和其它的原代细胞。此法已成功地用于研究膜的插入和移除的配体门控离子通道和G蛋白偶联受体，并评估变化贩卖由于受体激动剂和调节剂的存在下进行。主要方面包括标签的适当定位到细胞外的位置，并进行控制，以确保增加的BBS（作为标准的其他记者，如GFP）在功能上是沉默的。对...

利用荧光再加α-银环蛇毒素研究受体定 ​​位和动态是率先在烟碱乙酰胆碱受体13-15，毒素的内源靶的研究。随后，最小BGT结合肽（BBS）的掺入已被用于研究贩卖既兴奋性和抑制性配体门控离子通道和G蛋白偶联受体2,10,16-21的。本次论坛为基础的技术提供的优势，用于贩运研究，如表面生物素的方法，活细胞的抗体标记的抗体与细胞外抗原表位，和光漂白（FRAP）后荧光恢复其他方法。在细胞表面生物素游离胺的共价修饰，以影响细胞活性的潜力。基于抗体的研究常常受到阻碍表面抗原簇或封盖，它可以改变拐卖事件。由于对FRAP S中的漂白步骤tudies，一个重要的问题是损害底层的细胞结构。一个额外的优点是，信息标记的构建体也可用于生化方法学与生物素偶联银环蛇毒素对驴受体贩卖。这种技术是很容易适用于细胞系和原代细胞。对于在细胞表达烟碱乙酰胆碱（胆碱）受体的使用，胆碱受体拮抗剂筒箭毒碱，必须使用整个协议所指示的。对未转染的细胞在缺乏筒箭毒碱的执行一个简单的表面BGT标签（等同于内吞协议T = 0的时间点），将提供内源性胆碱受体的证据。 使用这种技术的一个重要的考虑因素是BBS的适当的插入，使得它存在于当目的蛋白被递送到质膜的细胞外的位置。例如，GABAAR亚基的N-末端结构域T​​RAF期间驻留在囊泡腔ficking并成为细胞外质膜受体插入后，使细胞表面的受体和其从细胞表面通过内吞事件去除的评估特异性标记。之前我们已经表明，除了GFP，myc和或BBS的表位GABAAR亚基这个领域在功能上保持沉默。标准控件应该执行，以确保标签的蛋白的表达水平相似，以不加标记的构建物，它适当地本地化的，并且它不影响受体的功能。转结构的这一特点也将有助于在故障排除过度担忧。

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			<td height="42" style="height:42px;width:227px;">Name of Material/ Equipment</td>
			<td style="width:201px;">Company</td>
			<td style="width:196px;">Catalog Number</td>
			<td style="width:187px;">Comments/Description</td>
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			<td height="20" style="height:20px;width:227px;">P3 Primary Cell 4D kit&#160;</td>
			<td style="width:201px;">Lonza</td>
			<td style="width:196px;">V4XP-3024</td>
			<td></td>
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			<td height="40" style="height:40px;width:227px;">&#945;-bungarotoxin, Alexa Fluor 647 conjugate</td>
			<td style="width:201px;">Molecular Probes</td>
			<td style="width:196px;">B35450</td>
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			<td height="40" style="height:40px;width:227px;">&#945;-bungarotoxin, Alexa Fluor 488 conjugate</td>
			<td style="width:201px;">Molecular Probes</td>
			<td style="width:196px;">B13422</td>
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			<td height="40" style="height:40px;width:227px;">&#945;-bungarotoxin, Alexa Fluor 594 conjugate</td>
			<td style="width:201px;">Molecular Probes</td>
			<td style="width:196px;">B13423</td>
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			<td height="20" style="height:20px;width:227px;">(+)-Tubocurarine chloride</td>
			<td style="width:201px;">Tocris</td>
			<td style="width:196px;">2820</td>
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			<td height="20" style="height:20px;width:227px;">mounting medium&#160;</td>
			<td>DAKO</td>
			<td style="width:196px;">cS703</td>
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			<td height="20" style="height:20px;">DPBS no calcium,magnesium&#160;</td>
			<td style="width:201px;">Invitrogen&#160;</td>
			<td style="width:196px;">14190-136</td>
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			<td height="20" style="height:20px;width:227px;">poly-D-lysine&#160;</td>
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			<td height="20" style="height:20px;width:227px;">gephyrin antibody</td>
			<td>Synaptic Systems</td>
			<td>147 011</td>
			<td>1:300</td>
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			<td height="20" style="height:20px;width:227px;">VIAAT/VGAT antibody</td>
			<td>Synaptic Systems</td>
			<td>131 002</td>
			<td>1:1000</td>
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			<td height="20" style="height:20px;width:227px;">anti GFP</td>
			<td style="width:201px;">Life Technologies</td>
			<td>A6455</td>
			<td>1:2000</td>
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			<td height="40" style="height:40px;width:227px;">glass bottom tissue culture dish</td>
			<td style="width:201px;">MatTek Corporation</td>
			<td colspan="2">P35G-1.5-14-C - Mattek Dishes</td>
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			<td height="40" style="height:40px;width:227px;">coverslips (round cover glass), #1 thickness, 12 mm</td>
			<td style="width:201px;">Warner Instruments</td>
			<td style="width:196px;">640-0702</td>
			<td></td>
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using an &#945;-bungarotoxin binding site tag to study gaba a receptor membrane localization and trafficking

这是越来越明显的神经递质受体，包括离子型GABA A受体（GABAAR），表现出高度动态的贩运和细胞表面的流动性1-7。为了研究受体细胞表面的定位和内吞作用，这里所描述的技术结合使用荧光​​α-银环蛇毒素与细胞表达含有α-银环蛇毒素（BGT）结合位点（BBS）的结构。在BBS（WRYYESSLEPYPD）是基于肌肉烟碱乙酰胆碱受体，其结合BGT以高亲和力8,9的α亚基。注册成立的BBS站点允许表面定位和受体插入或移除与应用外源性荧光BGT的测量，如以前在GABAA和GABAB代谢的跟踪受体2,10描述。除了 ​​信息的网站，我们氨基酸4与5的成熟GABAAR亚单位通过标准米之间插入一个pH敏感的绿色荧光蛋白（pHGFP 11）olecular生物学和PCR克隆策略（参见图1）12。 BBS的是3&#39;的pH值敏感的GFP报告，由13个氨基酸丙氨酸/脯氨酸接头隔开。对贩运这一出版物是基于固定样本中描述的研究中，pHGFP作为总的记者标记GABAAR亚单位蛋白水平，使BGT正常化标记受体的人口占总人口的受体。这最大限度地减少细胞与细胞的标记GABAAR亚基的较高或较低的基线表达所得BGT染色信号的变异性。此外，pHGFP标签可方便识别构建表达细胞的活的或固定成像实验。

一个信息标记构建体的表征，包括重要的控制，如确定所表达的蛋白质正确装配（特别是与受体的多个亚基组成），贩卖到细胞表面，并适当地局部化。抑制性突触是由γ-氨基丁酸的共定位该具有抑制支架蛋白gephyrin并结合在突触前的抑制端子，确定囊泡抑制性氨基酸转运加载GABA和甘氨酸进入突触小泡（VIAAT / VGAT）A受体表面的集群。 如图2顷 α2pHGFP+ BBS表达神经元与表面GABAAR标记BGT，...

Watch this Scientific Journal Video about Using an ÃŽÂ±-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking at JoVE.com

Using an ÃƒÅ½Ã‚Â±-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

使用α-银环蛇毒素结合位点标记来研究GABA A受体膜定位和贩运

在这里，我们展示了使用荧光染料的Alexa耦合到α-银环蛇毒素测定GABA A受体表面的定位和内吞作用的海马神经元。通过采用轴承结构结合α-银环蛇毒素，可以实现质膜蛋白的内吞贩运分析短外标签。

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Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

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大脑切片生物素化：一个<em&gt;前体内</em&gt;方法来衡量地区特有的质膜蛋白贩运成年神经元

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

通过RNA定位蝗虫天线中的气味受体基因<em&gt;原位</em&gt;杂交

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

用反褶积显微镜观察小鼠原发性下丘脑神经元 GLUT4 蛋白的动态图像

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

利用体外大鼠脑切片研究变性主要事件的一种替代方法

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

A High-content Assay for Monitoring AMPA Receptor Trafficking

一种用于监测 ampa 受体贩运的高含量检测方法

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different ...