这项工作导致本出版物已收到的资金从居里夫人COFUND节目"U-移动"，由马拉加大学和欧盟第七框架计划（FP7/2007-2013）根据GA编号246550共同出资。在我们实验室工作的支持，赠款从美国国立卫生研究院，美国农业部/ NIFA，美国国家科学基金会，BARD和BSF到VC

1。蛋白表达<ol><li> 表达系统的选择 选择系统，即向量和向量配送方式，最适合在特定的模式生物/细胞目的蛋白的表达。注意，我们的测定需要在容易可检测的量，这是最好的大批细胞的瞬时转化取得的测试蛋白质的表达。在植物中，例如，这是一个使用二进制质粒作为表达载体和农杆菌作为输送系统的最佳实现。 </li><li> 二元表达载体的构建 克隆感兴趣的蛋白（S）的编码序列（次）整合到表达载体单独或翻译融合到表位标签。使用载体适合于所选择的表达系统和标准分子生物学程序，用于基因的克隆。农杆菌介导的基因传递，采用二进制向量和介绍他们，也使用STANDA第三届协议，到农杆菌菌株，如EHA105，为后续接种植物组织。 </li><li> 植物种类的选择 选择其中感兴趣的蛋白（s）将待表达的植物物种。需要注意的是，虽然任何植物物种易被农杆菌介导的遗传转化，可以使用我们的植物种类的选择是本氏烟 ，这是容易生长，很容易受到土壤杆菌，并具有大叶这很容易进行接种。 </li><li> 植物生长 在与Pro-BX混合锅的锅（20厘米×20厘米×20公分）长天环保控制的条件（例如，生长室）130（即，16小时下生长一个工厂为4〜6周-150μEM-2 s-1的光在23°C和8小时黑暗，20°C）和40-65％的相对湿度。 1.5.2施肥偶尔与每个制造商的说明可商购的产品。一旦植物生长，选择叶与50毫米×70毫米或更大的尺寸（这些长度的测量不包括叶柄）农杆菌接种。 </li><li> 接种农杆菌 生长的农杆菌菌株携带二元构建体从步骤1.3过夜，在28℃在YEP培养基（1％蛋白胨，1％酵母提取物和0.5％NaCl）中补充有合适的抗生素（例如，100毫克表示被测蛋白（S）/ L链霉素和10mg / L利福平为pPZP-RSC2为基础的载体24-25）。 1.6.2离心细胞，再悬浮到OD 600 = 0.5的缓冲液浸润[10毫摩尔MgCl 2，10 mM的MES（pH值5.6），100μM的乙酰丁香酮]，并孵育在室温下2小时。渗透培养成叶子的背面侧，使用1毫升无针头的注射器;注意，叶片接种在原地，而附着到植物。 1.6.3浸润后，72小时16小时130-150＆光制下成长的植物＃181，E M-2 s-1的光在23℃/ 8小时黑暗，在20℃下收获前。 </li><li> 蛋白酶体抑制剂的应用 为支持该测试的蛋白质（s）是通过蛋白酶体途径降解的概念，使用特定的蛋白酶体抑制剂MG132和乳胞素28-29，这应该减少或什至阻止降解。前4小时收获（见步骤2.1），渗透与10μMMG132（EMD Millipore公司）或10微米乳胞素（Sigma-Aldrich公司）或农杆菌接种叶面积模拟处理与各自的溶剂，即0.1％DMSO叶水或蒸馏水。 </li></ol> 2。的无细胞提取物的制备<ol><li> 叶收获 收获叶的接种区域，通常约。 200-400毫克的鲜重，并磨成细粉末在液氮中。另外，珠击败组织，使用任何珠打浆机（例如，Biospec）或牙科混汞（比如TPC高级<html>技术）直接在退化缓冲区（见步骤2.2）。 </li><li> 蛋白提取 通过放置在地上组织成500μL的降解缓冲液制备总蛋白提取物[的50mM的Tris-HCl（pH为7.5），100 mM氯化钠，10毫摩尔MgCl 2，5mM的DTT，5mM的腺苷5&#39;-三磷酸，和1x蛋白酶抑制剂混合物（Sigma-Aldrich公司）。注意的是，蛋白酶抑制剂混合物主要影响丝氨酸，半胱氨酸，天冬氨酸和金属蛋白酶和不与26S蛋白酶干扰。由两个连续的离心澄清提取物以12,000×g离心5分钟。 </li><li> 蛋白质的降解反应 转让提取物，通常是20μL等体积，以离心管，并培育它们在室温下为增加的时间段。通常情况下，采样时间为零，5，10，15，20，和30分钟的时间点。停止反应，通过在SDS凝胶上样缓冲液煮沸，并用免疫印迹分析它们。 </li></ol>jove_title"&gt; 3。通过免疫印迹法检测蛋白质的降解<ol><li> 凝胶电泳 3.1.1解决的蛋白样品通过SDS-聚丙烯酰胺凝胶电泳并根据标准协议30电转移的解决蛋白至硝酸纤维素膜。 3.1.2测定蛋白浓度用Bradford法（Bio-Rad公司），并装载50-80元车道总蛋白微克。确保被同样加载的所有样本。用于装载控制，比较一个无处不在的蛋白质种类，比如哪些迁移，与大约50 kDa的一个相对电泳迁移率的一个主要带推定的Rubisco大连锁的强度，它们可以在考马斯蓝染色的凝胶上或丽春红S-或待检测荧光SYPRO Ruby的染色硝酸纤维素膜。 </li><li> 闭塞 阻挡膜用5％脱脂牛奶在TBST（10mM的Tris-盐酸，140 mM氯化钠，0.05％吐温20，pH7.4）中，在室温下1小时。 </l我&gt; <li> 一抗 在1％脱脂乳中的TBST稀释的初级抗表位的抗体，以制造商推荐的浓度和孵育的阻断膜在室温下1小时或过夜，在4℃下缓慢搅拌。 </li><li> 漂洗 漂洗膜漂洗20毫升TBST 15分钟一次，两次，持续5分钟，在室温下轻轻搅拌。 </li><li> 二抗 稀释的第二抗体（例如，抗兔IgG）在1％脱脂乳的TBST中偶联有辣根过氧化物酶（HRP），为制造商推荐的孵育与膜在室温下温和搅拌1小时。 </li><li> 发现 再次冲洗膜按照步骤3.4中所述。在TBST中的最后冲洗后，可视化所感兴趣的蛋白与辣根过氧化物酶化学发光底物，最常用的用ECL试剂盒。 </li></ol>

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没有利益冲突的声明。

该测定依赖于植物组织中的测试蛋白质的表达，因此，潜在的蛋白酶体降解过程显然已经发生的活体组织内。然而，我们测定蛋白质不稳定，仅在提取物，用时间为零的样品作为初始参考点。因此，我们将其定义为不含细胞的测定法。 对于该测定法的成功的一个重要的方面是正确的选择从其中被测试的蛋白质（s）将制备的表达载体的构建。除可对供试蛋白特异性抗体，它应该是表位标记进行检测免疫印迹。被标记的蛋白质应该从农杆菌二元载体来表达。尽管许多这样的载体可26，我们建议使用我们的模块化PSAT质粒系统24，其允许表达盒一步法插入pPZP-RCS2二元质粒衍生的载体24-25。该PSAT系统的另一个优点是，它允许从同一载体25，这是实验以测定一些蛋白酶底物或研究测试基板的交互件的影响，尤其是有用的多个基因的表达;用于农杆菌 VirD5的例子的共表达蛋白质与从蛋白酶体降解22 virF等保护蛋白酶体基板virF等结果进行交互。重要的是，扩大PSAT系列矢量已经包含了表位标记27的载体。不管所选择的克隆策略的，编码感兴趣的表位标记的蛋白质的序列应在二元载体用于随后的表达在植物组织中引入。因为该测定中使用的瞬时表达，二元载体不必包含选择标记用于稳定转化，但它们的存在不是有害的测定效率。 虽然是用多基因表达载体最好达到几个感兴趣的蛋白的共表达，它也可以由两个或三个农杆菌菌株，其中每一个带有一个独立的二元构建体的液体培养物的等量组合完成的。 另一个关键点是，在许多情况下，蛋白酶体降解测定法不仅蛋白酶底物，同时也对SCF传导途径的组分的表达涉及。例如，实验目的可能是要测试是否一个F-box蛋白识别和目标降解特定的底物。在这种情况下，这个表位标记的基板应共表达与未标记的F-box蛋白。 对数据的更详细的分析，蛋白质降解的程度可以容易地通过测量西方信号的相对强度定量Ú唱了最新的ImageJ软件（NIH）31。当执行这样的量化，它不是过度开发的印迹，以避免信号饱和度是很重要的。数据量化还要求所有的样品都在相对于它们的蛋白质含量为蛋白质的降解在SDS-聚丙烯酰胺凝胶上同样在该测定是通过减少在适当的蛋白条带的强度进行检测。这是通过测定每个细胞提取物中的蛋白质含量，并通过载荷相等各车道的（见步骤3.1）的后续验证实现。此外，在任何给定的时间过程实验，将所有样品应取自同一批提取物衍生的，有助于确保相等的加载。 对于这里描述的蛋白酶体降解的无细胞测定法使用植物作为一个实验系统。然而，在相同的实验设计，可用于任何其它有机体。该测定也可以通过弗思延长呃专注于SCF途径。例如，可以使用显性负对SCF组分CULLIN1（CUL1 DN）的形式来证明蛋白质降解对SCF依赖性机制。具体地，拟南芥CUL1的突变体与位置1和420之间被删除的氨基酸残基已被证明与SCF复合物的Skp1/ASK1组件进行交互，但不能与RBX1，它代表SCF 22的催化核心。抑制或减少底物降解的下列CUL1 DN的共表达表明对SCF途径的参与。 本试验利用蛋白质的利益表达在植物组织中，在自然的环境。一种可以替代的方法是将纯化重组蛋白，将它们添加到植物提取物，并按照它们的降解受到西方分析32。我们不建议这种战术的首选方法，因为重组蛋白做不永久š忠实地反映原生生物特性，但是，如果在植物中的表达是不可行的，使用重组蛋白的一定代表一个有价值的替代方法。

在泛素蛋白酶体途径正在成为一个普遍的机制，不同的生物反应，包括转录调控，细胞周期进程和信号转导，受体下调或胞吞作用，其中包括处理1-4的控制权。在此途径中，靶蛋白被标记由泛素，它们通过硫酯键的第一个连接到泛素激活酶E1，然后易位到的泛素缀合的酶E2的半胱氨酸氨基酸残基的残基;最后，E2具有泛素连接酶E3的交互，导致蛋白质底物的泛素化。最终，该多聚泛素蛋白被26S蛋白酶体识别并降解。在这种机制下，在E3酶指定的基板，并作为泛素蛋白酶体系统（UPS）的关键调控元件。 E3连接酶可以独立地采取行动，如RING结构域连接酶，或作为一个多亚基SCF（S部分kp1/Cullin/F-box蛋白）复合物，如F-盒结构域连接酶。 SCF-介导的蛋白酶体降解途径中涉及转录，细胞周期，信号转导5-10和许多其他主要的细胞功能的调节。 除了在细胞过程的调控这些关键角色，UPS采取中心舞台在许多植物 - 病原体相互作用。例如，越来越多的证据表明，一些植物病原体，包括根癌农杆菌，依赖于宿主的UPS，以促进感染过程11。土壤杆菌引发的植物，其中表示它的天然宿主的肿瘤生长，并且它也可以改变一个范围广泛的其他真核生物中，从真菌1,2对人类细胞12,13。在其感染时，根癌农杆菌出口的DNA元件（T-DNA）和几个毒力（虚象）的蛋白质进入宿主细胞12-13。这些蛋白质之一是virF等，找到的第一个F-box蛋白通过原核基因组14进行编码。由于SCF泛素连接酶复合物，virF等，其功能主机同源VBF 15的一部分， 通过 UPS介导的蛋白质降解，这大概是有利于入侵细菌的T-DNA的脱壳从与之配套的细菌和宿主蛋白，性virE2利于农杆菌感染和VIP1，分别为16,17。有趣的是，许多F-box蛋白，包括virF等，是由于自身蛋白酶解，这是由自身泛素化活性的18,19或由其他E3连接酶的量的F-box蛋白可以用作基板20-23介导的内在的不稳定。 当研究F-box蛋白，其它泛素连接酶，和/或它们的底物的生化活性，这将是非常有用的，采用一个简单而可靠的测定蛋白酶体降解。在这里，我们描述了一个这样的协议，用于分析在细胞蛋白质的稳定性- 免费的系统。在该测定中，UPS基板的稳定性在蛋白酶体降解途径中，诸如F-box蛋白，在无细胞系统的主要部件之一的存在或不存在进行了分析。一般来说，我们表示测试蛋白（S）在植物组织中，从这些组织准备的无细胞提取物和监测免疫印迹感兴趣的蛋白（S）的款项。蛋白降解的UPS依赖性机制被证明通过包含特定的蛋白酶体抑制剂和/或使用的一个SCF成分，卡林的显性负形式的共表达。而我们使用拟南芥VIP1 17蛋白的蛋白酶体降解的F-box蛋白VBF 15示出该试验中，它可被用来考察的其它任何蛋白酶底物的稳定性。

<table border="0" cellpadding="0" cellspacing="0" width="662">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Protein assay kit</td>
			<td style="width:129px;">Bio-Rad</td>
			<td style="width:119px;">500-0001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Proteinase inhibitor cocktail&#160;</td>
			<td style="width:129px;">Sigma-Aldrich</td>
			<td style="width:119px;">S8820</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Mini-Protean system</td>
			<td style="width:129px;">Bio-Rad</td>
			<td style="width:119px;">165-8000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Semi-dry western blotting SD electrotransfer system</td>
			<td style="width:129px;">Bio-Rad</td>
			<td style="width:119px;">170-3940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Affinity Purified Rabbit Anti-Ha</td>
			<td style="width:129px;">icllab</td>
			<td>RHGT-45A-Z</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Goat anti-Rabbit IgG Peroxidase Conjugate</td>
			<td style="width:129px;">Thermo Scientific</td>
			<td style="width:119px;">31460</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">BioTrace, NT nitrocellulose transfer membrane</td>
			<td style="width:129px;">Pall Corportation</td>
			<td style="width:119px;">27377-000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Immobilon western chemiluminescent HRP substrate</td>
			<td style="width:129px;">EMD Millipore</td>
			<td style="width:119px;">WBKL S0 050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">MG132</td>
			<td style="width:129px;">EMD Millipore</td>
			<td>474790-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Lactacystin</td>
			<td style="width:129px;">Sigma-Aldrich</td>
			<td>L6785</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Thermo Scientific Pierce Fast Western Blot Kit, ECL Substrate</td>
			<td style="width:129px;">Pierce</td>
			<td style="width:119px;">35055</td>
			<td></td>
		</tr>
	</tbody>
</table>

assaying proteasomal degradation in a cell-free system in plants

泛素 - 蛋白酶体途径降解蛋白质已经成为了一个广泛的细胞功能的频谱调控几乎所有真核生物中最重要的机制之一。具体而言，在植物中，泛素蛋白酶体系统（UPS），调节蛋白质降解和显著到的广​​泛的过程，包括免疫应答，开发和程序性细胞死亡的发展做出贡献。此外，越来越多的证据表明，许多植物病原体，如农杆菌 ，利用主机的UPS进行有效感染，强调的UPS在植物-病原体相互作用的重要性。 UPS的底物特异性是由E3泛素连接酶，其作用在音乐会与E1和E2连接酶识别和标记通过附加到它们的泛素分子链去往降解特定蛋白质分子来实现。一类的E3连接酶的是SCF（SKP1 / C阿林/ F-box蛋白）复合物，其特异性识别UPS的底物，并通过它的F-box蛋白组分瞄准他们对泛素化。调查的UPS中的感兴趣的生物过程中的潜在作用，设计一种简单，可靠的测定为UPS介导的蛋白质降解是重要的。在这里，我们描述了使用植物无细胞系统中的一个这样的测定。此法可以适用于不同的细胞过程调节的蛋白质降解的作用的研究，特别注重对F-box蛋白底物的相互作用。

图1中 ，用于从Zaltsman 等人 17，示出了用于检测蛋白酶体降解的无细胞系统代表实验。具体来说，我们通过 SCF VBF通路在全展示出一种植物防御相关蛋白VIP1由VBF F-box蛋白的不稳定本塞姆氏 。拟南芥VBF和HA标记VIP1（HA-VIP1）的蛋白质瞬时共表达，以及表达叶提取物中的HA-VIP1含量蛋白免疫印迹分析。这一分析表明，VIP1金额减少至一个显著程度，与VBF共表达，但在没有VBF共表达（ 图1A）保持相对稳定。需要注意的是稍微减少而不VBF的VIP1含量可能是由于内源烟草VBF活性水平低。 VIP1不稳定的VBF通过蛋白酶体降解机理是从它的我推断nhibition由MG132（ 图1A）。结果在图1A的定量由VBF，这是阻止MG132（ 图1B）表明几乎完全（≥90±5％）VIP1。注意在MG132的存在略高的水平VIP1的最有可能是通过抑制内源性VBF活动17解释。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51293/51293fig1highres.jpg" src="/files/ftp_upload/51293/51293fig1.jpg" /> 图1。VBF VIP1促进蛋白酶体降解HA-VIP1被单独表达或N。共表达与VBF 本塞姆氏叶。由此产生的蛋白提取物孵育指定的时间周期，并使用抗HA抗体免疫印迹分析。推定的Rubisco酶大连锁作为负载控制（ 一 ）。 Quantifieð印迹信号被表示为在温育期的开始在没有VBF所获得的信号的百分比。该数据代表与表示标准偏差（B）三个独立实验的平均值。这个数字是改编自Zaltsman 等 17

Watch this Scientific Journal Video about Assaying Proteasomal Degradation in a Cell-free System in Plants at JoVE.com

Assaying Proteasomal Degradation in a Cell-free System in Plants

有针对性的蛋白质降解是一个重大的监管机制，细胞的功能。它发生通过一个保守的泛素-蛋白酶体通路，其高度多聚泛素链的靶蛋白，然后作为分子的"标签"为26S蛋白酶体。在这里，我们描述了用于蛋白质的蛋白酶体降解的简单和可靠的无细胞测定法。

测定蛋白酶体降解的植物无细胞系统

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular ...

assaying-proteasomal-degradation-in-a-cell-free-system-in-plants

Nanyang Technological University

Research

JoVE Journal

Biology

14.3K 次观看.  Stony Brook University, State University of New York. 有针对性的蛋白质降解是一个重大的监管机制，细胞的功能。它发生通过一个保守的泛素-蛋白酶体通路，其高度多聚泛素链的靶蛋白，然后作为分子的"标签"为26S蛋白酶体。在这里，我们描述了用于蛋白质的蛋白酶体降解的简单和可靠的无细胞测定法。 

视频: Assaying Proteasomal Degradation in a Cell-free System in Plants

Maintaining Wolbachia in Cell-free Medium

In this video protocol, procedures are demonstrated to (1) purify Wolbachia symbionts out of cultured mosquito cells, (2) use a fluorescent assay to ascertain the viability of the purified Wolbachia and (3) maintain the now extracellular Wolbachia in cell-free medium.  Purified Wolbachia remain alive in the extracellular phase but do not replicate until re-inoculated into eukaryotic cells.  Extracellular Wolbachia purified in this manner will remain viable for at least a week at room temperature, and possibly longer.  Purified Wolbachia are suitable for micro-injection, DNA extraction and other applications. 


This video describes a method for purifying Wolbachia pipientis from an Anopheles gambiae cell line and then culturing the endosymbiont in cell-free medium. An assay for viability of the bacterium is demonstrated.

维持细胞培养基的Wolbachia

In this video protocol, procedures are demonstrated to (1) purify Wolbachia symbionts out of cultured mosquito cells, (2) use a fluorescent assay to ...

科研

生物学

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

焦磷酸测序：一个准确的基因分型的简单方法

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi筛选识别胚后的表型 C。线虫</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

含量测定的LRRK2的激酶活性在体外</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

协议用于实现<em&gt;大肠杆菌</em&gt;基于TX-TL无细胞表达系统合成生物学

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3. Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, &beta;-catenin. Two different methods are described to assess &beta;-catenin protein degradation in Xenopus egg extract. One method is visually informative ([35S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess &beta;-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of &beta;-catenin degradation.

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

重组β-catenin的降解<em&gt;爪蟾</em&gt;卵提取物

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

单通道细胞贴附膜片钳记录

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

测定蛋白酶体降解的植物无细胞系统

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含量测定的LRRK2的激酶活性在体外

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

协议用于实现

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

重组β-catenin的降解

单通道细胞贴附膜片钳记录