This work was supported by the National Institute on Aging (AG039768) to MHK. We thank Drs. Thomas Sharkey and Honggao Yan for generously providing the instruments, as well Sean Weise and Yan Wu for technical assistance.

1.试剂的配制<ol><li>制备聚合缓冲液（20mM的Tris，pH值7.4，100mM的氯化钠，1mM EDTA）中。存储在室温下，稳定了几个月。补充使用前1 mM的二硫苏糖醇（DTT）。 注：一个HEPES基缓冲液（10mM HEPES，pH为7.5，0.1毫摩尔EDTA，5毫DTT）也产生在tau蛋白聚集了类似的结果。 </li><li>制备硫磺素T或硫磺素的股票溶液（3毫米，溶解在聚合缓冲液中），并过滤通过0.22μm的无菌过滤器单元。储存在-20℃下在覆盖的铝箔，稳定数月的管。 </li><li>制备肝素原液（300μM，溶解在聚合缓冲液）。储存在-20℃下，稳定数月。 </li><li>准备二硫苏糖醇（DTT）的股票（1M，溶于水）。分装成1.5毫升管。存储在-20℃。前聚合测定法，解冻在RT的1M溶液。从这个1000倍的股票，准备100毫米的工作股票用去离子水等分。离开冰直到准备。 </li><li> -80去除头℃的冰柜。冰上解冻。调整头至规定浓度与聚合缓冲区。旋在微量在20800×g离心10分钟，在4℃以除去预先形成大的聚集体。该预纺丝步骤增加一致性每批的蛋白质制备的随后荧光测定。将上清液转移至另一管中;离开冰上，直到准备组装聚集反应。 </li></ol> 2.无染，终端分析注：该测定的聚合反应是在不存在的荧光染料进行。混合所有组分之后，使反应进行到预定的时间点。等分试样然后取出聚合反应，并用的ThT或THS为淀粉样蛋白的荧光读数之前结合混合。聚合反应的初始体积取决于所需要的时间点的数量。这种方法可能requIRE大量tau蛋白，但是快速，简单，并且可以在荧光计或一个多孔板读数器（见讨论）来实现。下面是一步一步的操作，使用ISA SPEX FluoroMax-2紧凑分光光度计进行荧光定量。 <ol><li>设置在1.5ml的Eppendorf管中的聚合混合物，如表1，每列代表所需的100微升反应，这是足够的一次性点测量的成分。调节量的基础上所需要的特定的实验时间点的整个聚合混合物。添加额外的10％的各组分给房间取样误差。此处示出的典型的含反应肝素可以通过花生四烯酸或聚集缓冲器被替换。添加DTT至1mM到反应混合物中。如果整个反应持续超过一天，补充新鲜的DTT每天（1毫米），以确保一个还原环境。 </li><li>反转管几次混合。解放军CE在一个37℃的恒温箱或水浴每个反应。搅拌不需要用于tau蛋白聚集。 </li><li>前测量荧光，在分光光度计打开（灯先，然后计算机）。 注：氙弧灯，可用于马上。然而，对于最佳的结果允许机器读取荧光之前预热约10分钟。 </li><li>启动计算机上的软件。 <ol><li>选择实时显示模式下仪表控制中心 ，设置激发波长为450纳米（狭缝到2纳米），发射波长为510纳米（狭缝5纳米）。关闭实时显示模式窗口，返回到仪器控制中心 。 </li><li>选择恒波长分析 ，按添加&gt;&gt;键上部框架添加波长集。 标准误差设置采集参数为1， 最大试验到3，然后单击添加 。 单击GO！打开数据显示窗口。 </li><li>在数据显示窗口中，单击开始ACQ打开新样品对话框 。选择“未知” 样品类型 。 </li></ol></li><li>以每100微升聚集混合，加入98微升聚合缓冲区和2微升3毫米硫黄素T.吸取几次，以混合。 </li><li>将整个混合物转移到反应杯（FCA3，外部尺寸，wxlxh = 12.5Hz毫米×12.5毫米×45毫米）。放置在样品室中的样品保持器的反应杯，并盖上盖子。单击运行收集荧光数据。记录数据。 </li><li>取出试管，倒出溶液。冲洗的反应杯由蒸馏水3次。干燥通过在与反应杯外送风。 </li></ol> 3.用染料，连续模式测定法上的SpectraMax M2酶标仪注：此试验不同于前一个，所述荧光染料的ThT或THS被包括在aggrega化反应。这允许相同的一组反应的连续测量。由于重复使用的反应，这种方法是更好地与一个自动多板读数器进行（如所示的SpectraMax M2的操作下）。一个普通的荧光也适用，但快速聚合反应测量的频率是比较有限的，由于操作的手动性质。 <ol><li>设置的聚集混合物在96孔板（96孔黑色实心板，以及体积360微升，平底），如表2，每列代表所需的200微升反应，这是足够的1的成分时间点测量。吹打几次拌匀。在整个实验过程中补充新鲜的1mM的DTT每一天。 </li><li>孵育96孔板在37℃。 </li><li>在荧光测定之前，每个时间点，打开多模式酶标仪和计算机。留出足够的时间马折角稳定，约10分钟。 </li><li>启动计算机上的软件。设定温度至37℃，并选择荧光强度（FI-顶读取）模式，设定激发波长450nm和发射波长在510纳米。 </li><li>插入的96孔板进抽屉，并按下READ键开始测量。 </li><li>看完后，取出板然后将其返回到37℃培养箱。数据复制并粘贴到Excel电子表格进行数据分析和绘图。 </li></ol> 4.染料，连续模式测定法上的紧凑型荧光光谱仪<ol><li>设置在1.5ml的Eppendorf管中的聚合混合物，如表3，每列代表所需的200微升反应，这是足够的一次性点测量的成分。 </li><li>反转管几次混合。 </li><li>打开分光光度计和软件设定为在步骤2.3和2.4。 </li><li>传送整个混合物吨OA比色皿。放置在样品室中的样品保持器的反应杯，并盖上盖子。单击运行收集荧光数据。记录数据。 </li><li>继续在适当的间隔通过点击运行并记录数据读取。如果聚合是在一个较高的频率（ 例如 ，每30或60秒），以进行监测，留在反应杯，并在机器的反应，直到测量完成时，或当有足够的时间来交换反应或比色皿。 </li><li>取出试管，倒出溶液。冲洗的反应杯由蒸馏水3次。干燥通过在与反应杯外送风。 </li></ol>

We have nothing to disclose.

该协议证明了不同的检测条件和检测磷酸化依赖的快速tau蛋白的聚集动力学的仪器。在终端测定中，荧光染料的ThT加到从主混合物在每个时间点除去反应的一部分。结合淀粉样蛋白诱导的荧光，然后测量26。在第二，与染料模式，tau蛋白聚集是在的ThT或THS的存在下，使这种类型的适合的tau聚集物的生长的实时自动评估反应。每种方法都有其优点和缺点。 终端模式反应?...

阿尔茨海默氏病（AD）是一个大的集合称为tau蛋白病的神经变性疾病中的一种。典型的病理Tau蛋白病的基本是神经原纤维缠结，在神经元，星形胶质细胞和小胶质细胞1-4。在NFT密度与认知功能障碍3,5和神经元丢失6。 NFT主要含有高度磷酸化tau蛋白（简称为“P-tau蛋白”此后），其形成直链或成对螺旋纤丝（PHF）7,8。头被认为是有利于轴突运输的是神经信号和贩运9,10必不可少的微管相关蛋白。每个tau蛋白分子含有2〜3磷酸盐在正常脑，而是由几个折叠的磷含量的增加在τ病变的患者11。多激酶可能有助于tau蛋白包括GSK3β（糖原合酶激酶3β）和CDK5（细胞周期素德依赖激酶5）12,13，但直接触发的病理磷酸化仍然遥遥无期14。异常磷酸化中或附近的微管结合基序上解离的tau从微管15，并且使头误定位到somatodendritic隔室，其中p-tau蛋白寡聚成直链或成对螺旋丝，可以最终聚合成NFT的内含物。 tau蛋白磷酸化，NFT形成与神经变性之间的紧密联系导致普遍的假设是p-tau蛋白缠结引起的凋亡和其他细胞毒性反应，因此是对神经变性τ病变16,17的根本原因。基于这个前提药物屏幕和早期临床试验已经启动18。然而，这种假设面临的挑战19,20。例如，圣克鲁斯等人表明，转基因小鼠的认知功能可以通过抑制突变体的表达来改进人类tau蛋白，即使NFTS继续形成从现有的tau蛋白分子21。在果蝇模型，NFT显示出螯合的有毒胞质tau蛋白以保护下面的神经元细胞22,23。显然，NFT的发病中的作用，如果有的话，将极大地影响τ病变治疗剂的发展方向。 在高浓度的，重组的或正常脑τ蛋白自发但慢慢地聚合成PHF状结构体外 ，通过几个β折叠优选荧光染料，电子显微镜和光散射光谱24-27的结合所指示的。添加肝素或花生四烯酸，丰富的脂肪酸在人类的大脑，大大加速了PHF形成头isoform-和诱导浓度依赖性28-32。有趣的是，tau蛋白纯化从AD大脑或体外磷酸化反应制得面面俱到一ggregates更快，更高效地26,33-35。这些结果与对 - tau蛋白的病理性作用良好的协议。 体外基于对tau的聚集系统可以因此用作一个强大的工具用于AD药物筛选。 定在发现的药物可控制的tau聚集在上升的兴趣的tau聚集和AD的渐进的神经变性之间的密切联系，以及在药物开发的最新故障靶向Aβ斑块，公元36-38的另一个重要的组织学标志物。事实上，几个小组已经在不同的通量开始药物屏幕，使用体外的tau聚集反应作为主要测定法。被发现的一些化学品的展示在体外 39-42对tau聚集的抑制或逆转的活动。但是，目前所有的tau蛋白聚集调节屏幕使用未经修改的头说错过荧光粉的主要病理标记ylation，提高用于在公元治疗使用这些化合物的特异性和功效的关注。 一个发展中的聚集试验的生化特性和AD药物筛选的主要障碍是生产足够数量的病理生理学相关tau蛋白的蛋白质。使用拉链辅助催化系统，其中tau蛋白的同种型1N4R和GSK-3β激酶共表达在大肠杆菌中的大肠杆菌亮氨酸拉链融合蛋白，我们已经克服了这个挑战（穗等人 ，提交;请参阅tau蛋白和P-tau蛋白的最终产品图1;另见43 P-tau蛋白的初步质谱鉴定）。从特异性针对tau的不同磷酸化位点9的抗体的面板，阳性信号出现在八个位置（数据未示出）。下面，我们描述的协议及仪器能够区分的聚集动力学ð未修改的tau蛋白和P-tau蛋白物种之间ifferences。这些检测是从公布的协议，可测得硫代黄素T（ThT的）或硫代黄素S（THS）的荧光的增加对淀粉样蛋白（tau聚集）结合26修改。在第一个“终端”，无染料的方法，聚合反应被组装并孵育在不存在淀粉样蛋白染料。在不同时间点，每个反应的等分试样被去除，并与含的ThT缓冲器的等体积混合，以阻止聚集和允许的ThT结合tau聚集。荧光是由IAP FluoroMax-2荧光计测量。在第二“与染料”持续监控测定法，或ThT的THS被包括在聚合反应。荧光可以连续地在整个实验手动测量或使用多板读数器。此外，我们描述了在连续测量莫使用的tau和对tau的一个近生理浓度的聚集的测定德。磷酸化的效果仍然易被察觉。下面，我们将描述一步一步的操作的程序，并显示这些分析的代表性结果。讨论一些每种方法的利弊，以及潜在的药物筛选的应用将随之而来。 以高浓度，tau蛋白聚集成淀粉样蛋白样结构自发。然而，在实验室中，tau蛋白纤维化典型地通过例如诱导肝素（平均分子量6000克/摩尔）和花生四烯酸加速。本文所示的例子包括30μM肝素。 tau蛋白的淀粉样蛋白聚集体的形成是通过从淀粉通过硫磺素T（ThT的）或硫磺素S（THS）结合而产生的荧光进行监测。结合后tau聚集，ThT的表现出红移荧光（激发：450纳米;峰值发射：485纳米）。 THS，而另一方面，具有弱发射在之前结合淀粉样蛋白为510nm（激发在450nm处），但这种fluorescencë增加显著中淀粉样蛋白的存在，如聚集的τ44。这两种染料的检测tau蛋白和P-tau蛋白聚集工作。因为的ThT（ 见图2）的强和相对宽的发光峰的，只有30在荧光单元减少在510nm％。为方便起见，我们使用的激发/发射波长（ 即 450纳米/ 510纳米）同样的组合来监控头聚集在使用任何染料。 头聚合所用的染料的存在或不存在下进行，这取决于测定的目的和τ蛋白的可用性。反应的两种模式如下所示。此外，我们证明了两种不同的仪器操作 - 单样本荧光计（ISA-SPEX FluoroMax-2）和多板酶标仪（Spectramax M2）。读者应该能够适应这些协议，以满足他们的特定需求和仪器的可用性。

<table border="0" cellpadding="0" cellspacing="0" width="449">
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		<tr height="64">
			<td height="64" style="height:64px;width:116px;">Name of the reagent</td>
			<td style="width:128px;">Company</td>
			<td style="width:103px;">Catalogue number</td>
			<td style="width:103px;">Comments</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:116px;">Trizma&#160;base</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:103px;">T1503</td>
			<td style="width:103px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:116px;">NaCl</td>
			<td style="width:128px;">Macron Fine Chemicals</td>
			<td style="width:103px;">MAL-7581-06</td>
			<td style="width:103px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:116px;">Ethylenediaminetetraacetic Acid (EDTA)</td>
			<td style="width:128px;">Invitrogen</td>
			<td style="width:103px;">15576-028</td>
			<td style="width:103px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:116px;">Thioflavin T</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:103px;">T3516</td>
			<td style="width:103px;">Stored in dark</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:116px;">Thioflavin S</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:103px;">T1892</td>
			<td style="width:103px;">Stored in dark</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:116px;">heparin</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:103px;">H3393</td>
			<td style="width:103px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:116px;">DL-Dithiothreitol (DTT)</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:103px;">D9779</td>
			<td style="width:103px;">Stored at 4&#176;C</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:116px;">96-well plate</td>
			<td style="width:128px;">Corning</td>
			<td style="width:103px;">3917</td>
			<td style="width:103px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:116px;">ISA SPEX FluoroMax-2</td>
			<td style="width:128px;">Horiba</td>
			<td style="width:103px;"></td>
			<td style="width:103px;"></td>
		</tr>
		<tr height="148">
			<td height="148" style="height:148px;width:116px;">SpectraMax M2 Multi-Mode Microlate Reader</td>
			<td style="width:128px;">Molecular Devices</td>
			<td style="width:103px;"></td>
			<td style="width:103px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:116px;">Mouse Anti-Tau Monoclonal Antibody</td>
			<td style="width:128px;">R&#38;D Systems</td>
			<td style="width:103px;">MAB3494</td>
			<td style="width:103px;">Stored at &#8211;80&#176;</td>
		</tr>
	</tbody>
</table>

in vitro aggregation assays using hyperphosphorylated tau protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

使用重组tau蛋白和对tau蛋白（ 图1），我们建立了两个不同的协议进行比较的tau和对tau的聚集的动力学，在结合到amyloidal蛋白聚集体采取的ThT和THS的强荧光发射的优点，其中包括头和对tau蛋白（ 图2）。具有或不具有在聚合反应中的荧光染料，我们通过磷酸化（ 图3-5）观测到的tau聚集的一致增强。这种刺激是独立肝素的（数据未示出）。减缓显著（ 图3?...

Watch this Scientific Journal Video about In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein at JoVE.com

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

未修饰的和磷酸化的tau蛋白以两种体外聚集测定中使用以显示磷酸化依赖快速聚合动力学。这些测定铺路用于可调节tau蛋白的倾向，以形成背后阿尔茨海默氏病的进展纤丝化合物未来画面。

体外聚集测定使用tau蛋白的蛋白质

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

in-vitro-aggregation-assays-using-hyperphosphorylated-tau-protein

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JoVE Journal

Biology

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

18.2K 次观看.  Michigan State University. 未修饰的和磷酸化的tau蛋白以两种体外聚集测定中使用以显示磷酸化依赖快速聚合动力学。这些测定铺路用于可调节tau蛋白的倾向，以形成背后阿尔茨海默氏病的进展纤丝化合物未来画面。 

视频: In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

科研

生物学

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions. Extracellular proteins, collagen I or von Willebrand factor are deposited within the microfluidic channel using active perfusion with a pneumatic pump. The matrix proteins are then washed with buffer and blocked to prepare the microfluidic channel for platelet interactions. Whole blood labeled with fluorescent dye is perfused through the channel at various flow rates in order to achieve platelet activation and aggregation. Inhibitors of platelet aggregation can be added prior to the flow cell experiment to generate IC50 dose response data.

The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions.

根据流量的血小板粘附和聚集，使用微流控流细胞

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

利用磁调制生物传感系统的生物检测的快速均质检测

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

雪旺的星形胶质细胞使用相互作用分析<em在体外检测

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

在活细胞中蛋白质聚集的4D成像

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

耦合检测监控蛋白复性<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

在体外细胞迁移和侵袭实验

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

在体外甲基化分析研究蛋白质精氨酸甲基化

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...