We thank Dr. Yuval Reiss and Dr. William Breuer for critically reviewing the manuscript and Omri Alfassy for helping in the development of the Ura3-GFP screen assay. We also thank Dr. M. Hochstrasser and Dr. R. Kulka for plasmids and strains. This work was funded by the Israeli Academy of Sciences (grant 786/08) and by the United States-Israel Binational Scientific foundation (grant 2011253).

1.细胞培养<ol><li>变换相应的营养缺陷型的酵母细胞，诸如Try467（ 表2），用含有（a）一种酵母URA3 -degron融合和（b）额外的代谢标记物的质粒的选择和维护的质粒。 注:合适的质粒的实例是YDpK-MET25p-Deg1-FLAG-Vma12-的Ura3（LYS2）（Deg1-紫外线）这是一个含有一种融合蛋白，其包含Deg1的酵母整合质粒的-从酵母转录得到的降解决定子。因子MAT2 17，FLAG表位-为检测通过免疫印迹法，Vma12 -稳定的ER蛋白，和的Ura3 15,18（ 图3A）。 （请参阅表3本研究和对合适的质粒的额外实施例中使用的质粒）。 </li><li>对于质粒的选择和维护，保持细胞在琼脂平板在合适的合成定义（SD）中缺少的氨基酸选择标记<s向上&gt; 19（例如，Deg1 -UV被选择并下的SD-LYS选择性培养基维持）。 </li><li>在对质粒保持一个合适的选择性的SD培养基上生长的细胞O / N在30℃下，直至稳定期为止。生长的细胞在试管中或在96孔板中，根据样本的数目要被检查（第2部分）。 </li></ol> 2.细胞制备方法分析<ol><li>当少量样品（N≤15）是待测定，稀释细胞并更换培养基如下: <ol><li>稀释50μl的细胞从一种O / N培养物用150微升每孔的SD培养基中，在96孔板（透明底）。注意，第一个样品被指定只作为空白包含介质。 </li><li>测定用酶标仪在96孔板中的样品的OD 600值。 </li><li>计算实际的OD 600在各孔中通过乘以OD 600读数乘以稀释因子（4个），从的whICH空白读数的值中减去。由0.55分，得到为1cm根据比尔 - 朗伯定律计算出的路径长度的归一化所得的值。注意，测量OD 600 200微升的细胞在标准的96孔板，当这个值是恒定的。 </li><li>计算出所需的用于获得的酵母细胞在量相当于OD 600为0.25，并将其转移到Eppendorf管中的准确体积。 </li><li>降速细胞以高速（12,000 xg离心），持续1分钟。 </li><li>弃上清，重悬细胞于1ml适当的选择性培养基（见3.1节），以获得OD 600为0.25。 </li><li>转移200微升稀释的菌株，以一个新的井在96孔板中。 </li></ol></li><li>当大量的样本（N&gt; 15）进行测试，稀释细胞并替换无需事先的OD 600测量的介质，具体如下: <ol><li>转让10微升静止细胞生长的O / N在96孔板到一个新的96孔板中含有合适的选择性培养基的190微升（稀释1:20）（见3.1节）。 </li></ol></li></ol>液体培养在选择性条件3.生长动力学（GILS） <ol><li>使用下面的媒体使用的Ura3，降解决定子记者成长的测量: <ol><li>对于非限定性条件下酵母菌的生长，利用质粒选择性SD存储介质的阳性对照（参见1.2节）。如果没有质粒的选择是必需的，例如，使用整合质粒如Deg1 -UV，含有所有必需氨基酸（SD完整）的SD培养基中时，可以使用。 </li><li>对于实验样品的测量限制条件下生长，使用SD-URA中。 </li></ol></li><li>设置一个多模酶标仪到:30℃，外径600测量的15分钟间隔的温育温度，1分钟每7分钟的轨道和线性振荡周期&lt;BR /&gt;注:此双模摇动优化，以获得细胞的均匀分布;然而，其他的晃动模式，甚至没有晃动可以正常工作。 </li><li>在孵育的细胞中，在30℃的微量板读数器的O / N或直到细胞达到稳定期（12-24小时）。 </li><li>原始数据导出到电子表格文件。 </li><li>确定细胞加倍的人口规模使用MDTcalc，即计算最小时间（小时）定制的软件程序，酵母生长动力学（最小的倍增时间（MDT）;有关详细信息，请参见第4节）。 </li></ol>最小的倍增时间4计算（MDT）:在MDTcalc算法的原理注:该MDTcalc算法的原理，使用在SD完全培养基中生长Try467酵母细胞（ 表2）的有代表性的样品，示于表1和图1中 ，示出了数据的电子表格和生长的地块，respectively。 <ol><li>减去的OD 600为空白值，在相应的孔获得，从每个样品中获得良好的读数。由0.55分所得的差异（对于96孔板），以校正所述光路长度。绘制的最后的值（ 表1中 ，反式。）对时间，以产生实际的酵母的生长曲线（OD 600 /小时）（ 图1A）。 </li><li>计算日志2（OD 600）为每个经变换的值以对数生长期的转化为线性曲线（ 图1B）。 </li><li>计算10的时间点的时间间隔（N = 10）开始于时刻0和移动一个时间点的时间，直到Ñ&lt;10的斜率（ 表1，斜率; 图1C）。 </li><li>计算每个斜率的倒数的值，以获得加倍所需的细胞群的时间（ 表1中 ，1 /斜率; 图1C）。计算最小1 /斜率值，以确定MDT的（ 表1，黑色矩形; 图1C）。提取所计算的MDT为从选定的时间间隔内的细胞样品中（ 表1中，1 /斜率，黑色矩形）（ 表1中，由红色矩形概述， 图1B，图1D）。 注:在SD-URA培养基中的细胞生长速率成反比的MDT。 </li></ol> 5.使用MDTcalc <ol><li>补充的程序的文件复制到在计算机中的指定文件夹。 </li><li>确保保存包含增长数据的电子表格文件，并关闭。打开MDTcalc应用程序，看看屏幕出现的开始。 <ol><li>插入所需信息，这表现在图2中: <ol><li>在"文件路径"，单击矩形上找到电子表格文件的权利。 </li><li>在"表名称"，其中t编写电子表格名称他的原始数据所在的位置。 </li><li>下"起始位置"中，插入的第一样本的时间0的电子表格坐标。 </li><li>下"时间列&#39;，插入信限定时间点列的位置（时间用秒来提供）。 </li><li>根据&#39;空白列&#39;，插入字母定义空白栏的位置（通常是但不一定是在A1孔在96孔板中）。 </li><li>在"OD值"，选择所需的MDT计算的开始（通常0.15-0.25）的最低转化OD 600值。设置此值可防止MDT的计算由于过度稀释培养的滞后期，这样MDT只为达到规定的OD 600值以上的样本计算的。 </li></ol></li><li>一旦最后的值被输入，按下屏幕底部的"计算"按钮。确保在正确的进度条逐渐变化到克颖。在计算过程中不要打开电子表格文件。 </li><li>导出的两组数据，可自动显示在屏幕上，在完成了的MDT计算到电子表格。确保该数据包括:（a）在各孔中传递的最小OD值测试（如在节5.2.1.6中设置）和（b）从所述的MDT来计算的时间间隔的起始时间点的MDT值（开始时间）。 </li></ol></li></ol>

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The authors have nothing to disclose.

在这里，我们描述了基于细胞的生长来确定蛋白质的相对降解率的测定，称为"液体培养基中的生长动力学下的选择性的条件"（GILS）。该GILS法有几个优点:它是简单的设置，数据采集和分析是很简单的，是非常模块化。因此，GILS可以同时应用于多个样品中，可适应于自动化高通量应用的一种用户友好的多井板格式。最重要的是，GILS提供高度可重复的，定量和可靠的数据和比选择为正/负生长的琼?...

泛素 - 蛋白酶体降解系统是一种主要的调节机，它有牵连的蛋白质稳态中的所有真核生物中的维持。 UPS的最初偶联物多泛素分子，以在此之后，多聚遍在蛋白标记的蛋白质被26S蛋白酶体降解的靶蛋白。在大多数情况下，速率限制为泛素介导的降解步骤是泛素化底物，由E2结合酶和E3结扎酶介导的（E3连接酶）1。因此，特定的蛋白质的细胞内稳定性反映了其易泛素缀合和它们的同源泛素酶的活性。 E3连接酶是UPS的主要底物识别元件。因此，这些内切酶识别它们的底物是不存在或是在其稳定的对应2不暴露于降解决定子。例如，许多的细胞周期调节米乌斯进行合成，并为了保持细胞周期进程，以便降低在时间上的具体方式。这些蛋白质的降解往往是由磷酸化控制，通过细胞信号调节的激酶介导的3,4。另一方面，异常折叠的蛋白质是通过隐蔽降解决定子的认可。这些是通常隐藏在天然结构并且在结构扰动被暴露的区域。这种降解决定子包括疏水区5-7和内在的无序段8。 由于泛素系统的精液发现对蛋白质降解和其基本特征中的网织红细胞裂解物9，酵母遗传学是有助的发现许多泛素系统10的组件。酵母作为模式生物的蛋白质降解的由UPS系统的分析的成功主要是由于这样的事实，所述UPS处于高LY保守的所有真核生物4，再加上他们的顺从作为一个实验系统。的确，基于酵母的系统通常用于破译泛素化机械的作用机制。 研究蛋白质降解的生物化学手段，通常需要准备的细胞提取物。虽然动物细胞的蛋白可以在相对 ​​温和的条件，同时保留蛋白质相互作用和功能下被提取，稳健的细胞壁中酵母11的存在需要相当苛刻的中断条件可能影响蛋白的恢复。事实上，不同的程序，酵母细胞破碎有很大的不同在他们恢复完整蛋白质在正确表示它们的相对丰度的细胞数量的能力。此外误差是固有的用于确定特定蛋白质的降解速率的不同方法:代谢标记为基础的"脉冲追踪"的实验，随后通过QQ，MSNmunoprecipitation，以隔离特定蛋白12，往往不是严格的定量。因此，当蛋白质的降解是由该方法相比，格外小心，应解释结果的行使。为了克服这个缺点，另一种放线菌酮（CHX）追法可聘请了12位。在此测定中，翻译抑制剂加入到细胞培养物和蛋白质的稳态水平的时间变化随后被监视。然而，放线菌酮的使用仅限于蛋白质具有相对短的半衰期（&lt;90分钟），由于长期抑制蛋白质的合成是细胞毒性的。值得注意的是，无论是在上述测定中需要使用蛋白质特异性抗体，这并不总是可用的。 为了克服这些技术限制，研究人员已开发了几种方法，不需要的细胞的提取和蛋白质直接处理。一种方法是基于建立营养缺陷型酵母的圣菌株，通过缺失基因编码的必需的代谢酶的获得。这样的基因包括HIS3，LEU2，LYS2和TRP1，对于所需的氨基酸生物合成的酶的编码，以及URA3，其编码的OMP脱羧酶（的Ura3）的嘧啶核糖核苷酸生物合成的必需酶。的ura3已被广泛用于蛋白质降解研究。在这些测定中，的Ura3的组成型表达拯救的ura3细胞的尿嘧啶缺陷型培养基中生长13。因此，通过降解决定子的融合不稳定的Ura3可以减少对基本培养基中缺乏尿嘧啶的细胞生长。此方法已被应用于各种蛋白质降解的研究，包括降解的识别决定因素5，E3连接酶14和辅助泛素化因子15和新颖的UPS降解决定子16的发现。所有的这些方法中使用的琼脂平板测定法读出的细胞生长。然而，T他生长标准（正/负增长），而健壮和有效的，主要是定性的，并且不提供用于评估降解决定子的效力或各种辅助降解因素的相对贡献重要的定量信息。 因此，我们开发和利用酵母载体和筛选方法的URA3 -degron融合系统使蛋白质降解系统和定量的分析。该协议基于一个易于手柄测定，其测量的生长动力学在选择性条件下（GILS）液体培养和对标准生长曲线的生成。酵母生长动力学的特征在于三个主要阶段 - 滞后，指数（日志）和固定相。计算过程中的选择性的条件下的对数期，这是由的Ura3，降解决定子的表达水平的测定酵母的复制动力学，提供了一个无偏定量测定öF蛋白的降解。这个方法可以适用于测量和比较同时在多种菌株和在各种条件下的多个UPS基材的降解速率。

<table border="0" cellpadding="0" cellspacing="0" width="790">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:184px;">Name of Material/ Equipment</td>
			<td style="width:149px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:339px;">Comments/Description</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:184px;">Difco yeast nitrogen base w/o amino acids and ammonium sulfate</td>
			<td>BD Biosciences</td>
			<td>233520</td>
			<td rowspan="6" style="width:339px;">For yeast growth on SD minimal media.&#160; Amino acids to be supplied are: Arginine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium sulfate</td>
			<td>Sigma - Aldrich</td>
			<td>A4418</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose</td>
			<td>Sigma - Aldrich</td>
			<td>16325</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Adenine</td>
			<td>Sigma - Aldrich</td>
			<td>A8626</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Uracil</td>
			<td>Sigma - Aldrich</td>
			<td>U0750</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Amino acids</td>
			<td>Highest purity available&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:184px;">96 well plates</td>
			<td style="width:149px;">Nunc</td>
			<td style="width:119px;">167008</td>
			<td>Any other compatible brand can be used</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:184px;">Cyclohexamide&#160;</td>
			<td style="width:149px;">Sigma - Aldrich</td>
			<td style="width:119px;">C7698</td>
			<td>Working conc. 0.5 mg/ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:184px;">Infinite 200 PRO series</td>
			<td style="width:149px;">Tecan</td>
			<td style="width:119px;"></td>
			<td style="width:339px;">For yeast incubation and OD 600 measurements. Any other compatible temp-controled reader can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">MDTcalc</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td>Experimental software</td>
		</tr>
	</tbody>
</table>

reporter-based growth assay for systematic analysis of protein degradation

通过泛素 - 蛋白酶体系统（UPS），蛋白质降解是一个主要的调节机制在所有真核生物蛋白的动态平衡。该标准方法测定细胞内蛋白质降解依赖于生化分析了以下的蛋白质下降的动力学。这样的方法通常是费力和费时，因此不适合进行实验，目的是评估多种底物和降解的条件。作为另一种选择，细胞生长为基础的分析已被开发出来，那是，在他们的常规格式，不能定量确定在蛋白质水平的相对变化终点分析。 在这里，我们描述了一种方法，通过它们连接到酵母细胞的生长动力学，忠实地决定改变蛋白质的降解率。该方法是基于已建立的选择系统，其中的URA3的尿嘧啶营养缺陷型-deleted酵母细胞是通过外源表达报告蛋白获救，包括必要的URA3基因和一个退化行列式（降解决定子）之间的融合。报告蛋白被设计成使得它的合成速率是恒定的，而其降解速率由降解决定子确定。作为细胞生长的尿嘧啶缺陷型培养基是成比例的Ura3的相对水平，生长动力学的完全依赖于报告蛋白降解。 这种方法精确测量变化的细胞内蛋白质降解动力学。它适用于:（a）评估称为泛素结合因子蛋白水解（B）的相对贡献E2结合酶结构与功能分析（三）鉴定及新型降解决定子表征。该degron- URA3基于系统的应用超越蛋白降解字段，因为它也可以适用于蛋白质水平与其他细胞途径功能相关联的监视更改。

调查记者衬底的降解Doa10途径的酶的作用 为了测试GILS方法与其相比较传统的降解测定法的有效性。该实验评估对ER-膜的组成部分的相对贡献本地化Doa10的E3连接酶复合物20,21的蛋白质量控制记者基板Deg1- VU（ 图3A）的降解。该Deg1- VU质粒整合到野生型细胞或进入细胞缺乏该基因的E2结合酶，UBC7（ubc7）和蛋白质的稳定?...

Watch this Scientific Journal Video about Reporter-based Growth Assay for Systematic Analysis of Protein Degradation at JoVE.com

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Here we describe a robust biological assay for quantifying the relative rate of proteolysis by the ubiquitin-proteasome system. The assay readout is yeast growth rate in liquid culture, which is dependent on the cellular levels of a reporter protein comprising a degradation signal fused to an essential metabolic marker.

记者的增长分析的蛋白质降解的系统分析

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard ...

reporter-based-growth-assay-for-systematic-analysis-protein

Research

JoVE Journal

Biology

10.6K 次观看.  The Hebrew University of Jerusalem. Here we describe a robust biological assay for quantifying the relative rate of proteolysis by the ubiquitin-proteasome system. The assay readout is yeast growth rate in liquid culture, which is dependent on the cellular levels of a reporter protein comprising a degradation signal fused to an essential metabolic marker.

视频: Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

科研

生物学

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

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 ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

视频人类胚胎干细胞克隆生长的生物信息学分析

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

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 ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

成长型测定和遗传需求生化确认的蛋白质降解<em&gt;酿酒酵母</em

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic ...

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein synthesis and protein degradation. Many assays reporting on protein abundance (e.g., single-time point western blotting, flow cytometry, fluorescence microscopy, or growth-based reporter assays) do not allow discrimination of the relative effects of translation and proteolysis on protein levels. This article describes the use of cycloheximide chase followed by western blotting to specifically analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast). In this procedure, yeast cells are incubated in the presence of the translational inhibitor cycloheximide. Aliquots of cells are collected immediately after and at specific time points following addition of cycloheximide. Cells are lysed, and the lysates are separated by polyacrylamide gel electrophoresis for western blot analysis of protein abundance at each time point. The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. The procedure may be used to investigate the genetic requirements for and environmental influences on protein degradation.

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Protein abundance reflects the rates of both protein synthesis and protein degradation. This article describes the use of cycloheximide chase followed by western blotting to analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast).

蛋白质降解的放线菌酮大通分析<em&gt;酿酒酵母</em

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein ...

Assays for the Degradation of Misfolded Proteins in Cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

测定法错误折叠的蛋白质在细胞中的降解

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. Arabidopsis thaliana-Agrobacterium tumefaciens provides an excellent model system to study bacterial pathogenesis and plant interactions. Previous studies of plant-Agrobacterium interactions have largely relied on plant cell suspension cultures, the artificial wounding of plants, or the artificial induction of microbial virulence factors or plant defenses by synthetic chemicals. However, these methods are distinct from the natural signaling in planta, where plants and microbes recognize and respond in spatial and temporal manners. This work presents a hydroponic cocultivation system where intact plants are supported by metal mesh screens and cocultivated with Agrobacterium. In this cocultivation system, no synthetic phytohormone or chemical that induces microbial virulence or plant defense is supplemented. The hydroponic cocultivation system closely resembles natural plant-microbe interactions and signaling homeostasis in planta. Plant roots can be separated from the medium containing Agrobacterium, and the signaling and responses of both the plant hosts and the interacting microbes can be investigated simultaneously and systematically. At any given timepoint/interval, plant tissues or bacteria can be harvested separately for various &#34;omics&#34; analyses, demonstrating the power and efficacy of this system. The hydroponic cocultivation system can be easily adapted to study: 1) the reciprocal signaling of diverse plant-microbe systems, 2) signaling between a plant host and multiple microbial species (i.e.&#160;microbial consortia or microbiomes), 3) how nutrients and chemicals are implicated in plant-microbe signaling, and 4) how microbes interact with plant hosts and contribute to plant tolerance to biotic or abiotic stresses.

The described hydroponic cocultivation system supports intact plants with metal mesh screens and cocultivates them with bacteria. Plant tissue, bacteria, and secreted molecules can then be separately harvested for downstream analyses, simultaneously allowing for the molecular responses of both plant hosts and interacting microbes or microbiomes to be investigated.

用于植物/微生物分子相互作用和信号的同时系统分析的水培共培养系统

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. ...

Precise, High-throughput Analysis of Bacterial Growth

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the systems level. Recent studies have reported correlations between bacterial growth and genome-wide events, such as genome reduction and transcriptome reorganization. Correctly analyzing bacterial growth is crucial for understanding the growth-dependent coordination of gene functions and cellular components. Accordingly, the precise quantitative evaluation of bacterial growth in a high-throughput manner is required. Emerging technological developments offer new experimental tools that allow updates of the methods used for studying bacterial growth. The protocol introduced here employs a microplate reader with a highly optimized experimental procedure for the reproducible and precise evaluation of bacterial growth. This protocol was used to evaluate the growth of several previously described Escherichia coli strains. The main steps of the protocol are as follows: the preparation of a large number of cell stocks in small vials for repeated tests with reproducible results, the use of 96-well plates for high-throughput growth evaluation, and the manual calculation of two major parameters (i.e., maximal growth rate and population density) representing the growth dynamics. In comparison to the traditional colony-forming unit (CFU) assay, which counts the cells that are cultured in glass tubes over time on agar plates, the present method is more efficient and provides more detailed temporal records of growth changes, but has a stricter detection limit at low population densities. In summary, the described method is advantageous for the precise and reproducible high-throughput analysis of bacterial growth, which can be used to draw conceptual conclusions or to make theoretical observations.

Quantitative evaluation of bacterial growth is essential to understanding microbial physiology as a systems-level phenomenon. A protocol for experimental manipulation and an analytical approach are introduced, allowing for precise, high-throughput analysis of bacterial growth, which is a key subject of interest in systems biology.

细菌生长的精确、高通量分析

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the ...

记者的增长分析的蛋白质降解的系统分析

摘要

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此视频中的章节

肌动蛋白共沉淀测定; F -肌动蛋白结合蛋白分析

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

视频人类胚胎干细胞克隆生长的生物信息学分析

膜蛋白的绿色荧光蛋白为基础的表达筛选

成长型测定和遗传需求生化确认的蛋白质降解

蛋白质降解的放线菌酮大通分析

测定法错误折叠的蛋白质在细胞中的降解

用于植物/微生物分子相互作用和信号的同时系统分析的水培共培养系统

细菌生长的精确、高通量分析

记者的增长分析的蛋白质降解的系统分析

摘要

探索更多视频

此视频中的章节

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

视频人类胚胎干细胞克隆生长的生物信息学分析

膜蛋白的绿色荧光蛋白为基础的表达筛选

成长型测定和遗传需求生化确认的蛋白质降解

蛋白质降解的放线菌酮大通分析

测定法错误折叠的蛋白质在细胞中的降解

用于植物/微生物分子相互作用和信号的同时系统分析的水培共培养系统

细菌生长的精确、高通量分析

肌动蛋白共沉淀测定; F -肌动蛋白结合蛋白分析