Name: determining cell-surface expression and endocytic rate of proteins in primary astrocyte cultures using biotinylation
Uploaded: 2017-07-03T15:00:00
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该项目由加拿大卫生研究所PG＃20R47867支持。

1.通过生物素化测定星形胶质细胞中的相对细胞表面蛋白表达注意：在这里，我们说明了这种生物素化技术在细胞外基质分子层粘连蛋白对透水通道水通道蛋白-4（AQP4）的细胞表面定位的影响的研究中的应用。该测定所需的专用材料包括磺基-NHS-LC-生物素和链霉亲和素 - 琼脂糖树脂（参见材料表 ）。 <ol><li>使用Noel 等人概述的方法12 ，在...

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修改： 由于这些方法设计用于贴壁细胞，我们已经规定了使用含有100mg / L MgCl 2· 6H 2 O的PBS和 100mg / L的CaCl 2 （CM-PBS）洗涤步骤，并作为某些缓冲液的碱基，以确保细胞保持附着于培养物表面，并且细胞 - 细胞结不被破坏。然而，如果细胞在程序的每个步骤之间沉淀，则该协议也可以应用于非贴壁细胞类型。在这些情况下，可以用PBS?...

细胞表面的蛋白质起着维持细胞功能的重要作用。在许多情况下，它们的活性依赖于或者通过内吞过程调节，这些内吞过程暂时将其封闭在胞内位点，或者将它们引导到降解途径1,2,3,4,5 。在这里，我们强调了2种基于生物素化的方法，旨在允许用户专门标记和分离在质膜上表达的蛋白质，以及新内化的蛋白质。通过这些方法，可以量化任何目的蛋白质的细胞表面表达和内吞速率，从而允许更清楚地评估其调节。 通过生物素化测定相对细胞表面蛋白的表达 生物素或维生素B7（以前称为维生素H 6 ）是一种小水溶性分子，可用于化学修饰生物分子的活性胺，巯基和羧基。目前的细胞表面生物素化试剂主要由生物素或其衍生物的膜 - 不渗水磺化N-羟基琥珀酰亚胺（磺基-NHS）酯组成，其与设在蛋白质赖氨酸残基侧链上的胺反应，当细胞表面在碱性条件下脱质子时，后者与生物素部分7形成酰胺键。因此，可以通过使用抗生物素蛋白（一种对生物素具有极大亲和力的66-69kDa四聚体蛋白）来分离细胞表面蛋白，以约10-15的解离常数结合后者，将其标记为最强非共价相互作用已知8 </sup&gt; ， 9 。 在以前的研究中已经使用了许多量化蛋白质在细胞表面的方法。使用针对目的蛋白质特异性的荧光标记抗体进行标记，然后通过荧光显微镜观察是常用的方法，但是严重依赖于可结合细胞外表位的抗体的可用性。最近，已经成功地使用涉及使用与暴露于酸性介质反应的具有pH敏感性荧光团的嵌合蛋白质的方法。然而，这样的测定通常涉及这些构建体在其中未被天然存在的目的蛋白质的细胞系中的外源表达。这些方法仍然能够提供关于亚细胞定位和胞外步行的有价值的信息因此如果这些工具可用，则应与本文所述的基于生物素化的方法结合使用。 在典型的生物素化测定中，首先在4℃PBS中彻底洗涤细胞。这样可以消除由培养基引入的任何痕量的血清蛋白，从而确保在下一步中这些不会消耗过量的生物素。更重要的是，降低温度会导致内吞作用显着减速。然后加入生物素化试剂。接下来，再次洗涤细胞，然后与含有甘氨酸或NH 4 Cl的淬灭缓冲液一起温育，其目的是灭活所有剩余痕量的未反应的生物素。然后将细胞裂解，随后加入琼脂糖固定的链霉抗生物素蛋白以沉淀生物素化的蛋白质。分析通常通过western印迹进行，允许各种pr的相对细胞表面表达oteins要量化。 由于该测定的基础，它仅适用于具有暴露于细胞外环境的部分的蛋白质。可能在其环区内可能具有许多反应性赖氨酸的多巴跨膜蛋白是该方法中最容易的，而单程蛋白往往不太容易被生物素化。即使在这些情况下，仍然存在构象变化或分子间相互作用可能堵塞某些活性位点的可能性，导致低于预期的生物素化产率。 通过生物素化测定细胞表面蛋白的内在化率 该测定的原理与细胞表面生物素化的原理大致相同，除了一些例外，其中最重要的是使用可逆生物素化试剂。生物素组（其中的）具有disu在其结构内的易受还原剂影响的粘合剂;这被开发以确保仅在测定期间摄入细胞内部位的细胞表面蛋白质将被遗传生物素化。测定通常以以下方式进行。首先用冷试剂洗涤细胞并生物素化，然后重新引入37℃的细胞培养基，将细胞返回培养箱;这导致标记的细胞表面蛋白质经历内吞。然后加入不能穿透膜的还原剂谷胱甘肽，以破坏与残留在细胞表面上的蛋白质连接的生物素部分的二硫键。最后，破坏的二硫键与碘乙酰胺反应，消耗不稳定的硫醇基并防止键重组。如前所述，然后将细胞裂解，并使用链霉抗生物素蛋白 - 琼脂糖沉淀标记的蛋白质。 限制光盘由于在方法之间共享的相似之处，上一节中所使用的内容也适用于此。此外，值得铭记的是，该测定中涉及的温度变化阻止了对于每次增量的时间内含量多少蛋白质的确切测定，特别是在迅速内化或快速回收蛋白质的情况下。因此，该测定仅提供了内吞率的半定量估计。全内反射荧光显微镜可用于跟踪每个加载的囊泡的摄取，并提供更精确的内吞动力学测量。因此，假设目标蛋白的荧光标记嵌合构建体可用，因此可以提供非常有用的补体。

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		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Ammonium chloride (NH4Cl)</td>
			<td style="width:219px;">Fisher Scientific</td>
			<td style="width:129px;">A661-500</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A9647-50</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Bromophenol blue</td>
			<td>Bio-Rad</td>
			<td>#1610404</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">cOmplete protease inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>11697498001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Disodium ethylenediaminetetraacetate dihydrate (EDTA)</td>
			<td>Bio-Rad</td>
			<td style="width:129px;">#1610729</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Dithiothreitol (DTT)</td>
			<td>Bio-Rad</td>
			<td>#1610611</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco/Thermo Fisher Scientific</td>
			<td>11960-044</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">EZ-Link Sulfo-NHS-LC-Biotin</td>
			<td>Thermo Fisher Scientific</td>
			<td>#21335</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">EZ-Link Sulfo-NHS-SS-Biotin</td>
			<td>Thermo Fisher Scientific</td>
			<td>#21331</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Fetal bovine serum</td>
			<td>Gibco/Thermo Fisher Scientific</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Glycerol</td>
			<td>Fisher Scientific</td>
			<td>BP229-1</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G8898&#160;</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Iodoacetamide</td>
			<td>Bio-Rad</td>
			<td>#163-2109</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane</td>
			<td>Sigma-Aldrich</td>
			<td>L2020</td>
			<td>Thaw on ice.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">L-glutamine</td>
			<td>Gibco/Thermo Fisher Scientific</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Mouse monoclonal anti-&#946;-actin antibody (AC-15)</td>
			<td>Sigma-Aldrich</td>
			<td>A5441</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Mouse monoclonal anti-&#946;-dystroglycan antibody (43DAG1/8D5)</td>
			<td>Leica Biosystems</td>
			<td>B-DG-CE</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Penicillin/streptomycin</td>
			<td>Gibco/Thermo Fisher Scientific</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Peroxidase AffiniPure Donkey Anti-Mouse IgG (H+L)</td>
			<td style="width:219px;">Jackson ImmunoResearch Laboratories</td>
			<td>715-035-150</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L)</td>
			<td style="width:219px;">Jackson ImmunoResearch Laboratories</td>
			<td>111-035-045</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Phosphate buffer saline</td>
			<td>Gibco/Thermo Fisher Scientific</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Reduced glutathione</td>
			<td>Sigma-Aldrich</td>
			<td>G6529</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Sodium chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma-Aldrich</td>
			<td>862010&#160;</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Sodium hydroxide (NaOH)</td>
			<td>Fisher Scientific</td>
			<td>S318-100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Streptavidin agarose resin</td>
			<td>Thermo Fisher Scientific</td>
			<td>#20347</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Rabbit polyclonal anti-AQP4 antibody</td>
			<td>Alomone</td>
			<td>AQP-004</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Tris base (Trizma base)</td>
			<td>Fisher Scientific</td>
			<td>BP152-1</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Tris-HCl</td>
			<td>Fisher Scientific</td>
			<td>BP153-1</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

determining cell-surface expression and endocytic rate of proteins in primary astrocyte cultures using biotinylation

细胞表面蛋白介导各种功能。在许多情况下，它们的活性受调节其在质膜上的水平的内吞过程的调节。在这里，我们提供了有助于研究这些过程的两种方法的详细方案，这两种方法均基于细胞表面蛋白的生物素化原理。第一个被设计为允许半定量测定细胞表面上特定蛋白质的相对水平。其中，细胞的质膜蛋白的赖氨酸残基首先用生物素部分标记。一旦细胞被裂解，然后可以通过利用琼脂糖固定的链霉亲和素通过利用后者对于生物素的天然亲和力来特异性沉淀这些蛋白质。然后可以通过标准的Western印迹方法分析以这种方式分离的蛋白质。第二种方法提供了确定特定细胞吞噬率的方法ar细胞表面目标在一段时间内。首先用含有可切割二硫键的生物素衍生物修饰细胞表面蛋白。然后将细胞转移回正常培养条件，这导致一部分生物素化蛋白质的内吞摄取。接下来，使用膜不可渗透的还原剂谷胱甘肽使非内化生物素基团的二硫键减少。通过这种方法，内吞的蛋白质因此可以以高度的特异性被分离和定量。

使用细胞表面生物素化来评估星形胶质细胞中AQP4的质膜表达 使用所述方法将层粘连蛋白处理的星形胶质细胞培养物和未处理的对照细胞进行细胞表面生物素化。用琼脂糖缀合的链霉亲和素沉淀生物素化的蛋白质，然后通过SDS-PAGE分离。探测细胞表面级分作为细胞表面加载对照的AQP4和β-dystroglycan（β-DG），同时将输入和细胞内级分吸入AQP4和β-肌动蛋白（ <...

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Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

本报告提供了两种基于生物素化的方法，设计用于测定在质膜上表达的蛋白质的细胞表面表达和内吞率。

使用生物素化测定原代星形细胞培养物中蛋白质的细胞表面表达和吞噬率

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at ...

Research

JoVE Journal

Neuroscience

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

嵌入后突触蛋白的免疫标记海马切片培养

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

科研

神经科学

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

细胞表面和内蛋白质的现场培养神经细胞的抗体后喂养差异化标记

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

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 ...

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

动物的基因分型快速其次是建立大脑神经元原代培养

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

测定冷灵敏度和适应小鼠一种简单廉价的方法

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

评估主要神经再生<em&gt;爪</em&gt;胚胎使用免疫染色

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During ...

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

生活多巴胺能神经元可靠的鉴定中脑培养使用RNA测序和TH-启动子驱动的EGFP表达

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

一种新的体外活体成像法测定星形胶质细胞的吞噬作用 pH 指标-共轭突

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major ...

In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films

In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films

在 vivosilk/aav 薄膜在光基因表达中的靶向表达

The quest to understand how neural circuits process information in order to drive behavioral output has been greatly aided by recently-developed optical ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

通过Sialoglycan的代谢标记识别主要神经干细胞和祖细胞的细胞表面标记

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...