的的质粒pCG001表达Ubp1好心提供的罗汉贝克在约翰·科廷学校的医学研究。这项工作是由of Dimes的研究资助，2011财年3月，3月Dimes的瓦西里·奥康纳入门学术研究奖＃5-FY20和美国国家科学基金会职业奖0746796。

1。应变工程工作流程<ol><li>插入序列编码（一）的膜定位序列，（b）一种快速成熟的荧光蛋白和（c）的蛋白酶识别序列的N-末端和在帧与感兴趣的蛋白质（ 例如，一个转录因子）。我们采用针对性的TSR-金星记者2膜，并融合到的蛋白酶识别序列的泛素（Ub）表达λ噬菌体阻遏CI蛋白质分子的数量进行计数。我们如何构建的融合蛋白TSR-金星-泛素CI，CI编码区取代了野生型和将构建到E.详细信息大肠杆菌染色体λRed重组3，详细描述在文献。 1。 </li><li>确认所有修改测序。 </li><li>变换特定于上面使用到的菌株表达的荧光报道和兴趣蛋白质的识别序列的质粒编码的蛋白酶在翻译融合。我们使用的的蛋白酶Ubp1，立即裂解后的C-末端泛素残基4。 </li></ol> 2。培养细胞和制备样品<ol><li>选择一个E.大肠杆菌菌落兴趣的新鲜条琼脂平板上为1毫升的M9媒体5辅以，与1X MEM氨基酸和适当的抗生素。在所需温度下在摇床上孵育足够长，达到OD 600&gt; 1.0（在600nm处的光密度）。 </li><li>稀释为1毫升新鲜的M9培养基中的文化与适当的抗生素OD 600 = 0.02。在适当的温度下，在振动机中孵育。如果需要的话，可以提高低温生长的初始细胞密度。 </li><li>当OD 600 = 0.2-0.3（在37℃下的初始细胞培养在OD 600 = 0.02，这将需要3-4小时），开始制备琼脂糖凝胶垫（步骤3）。 </li><li>该细胞是准备用于成像当OD <suB&gt; 600 = 0.3-0.4。 </li><li>转移至1.5 ml离心管中，离心分离机在台式的离心10000×g离心1分钟1毫升孵育文化。 </li><li>弃上清，加入1毫升新鲜的M9培养基中，并轻轻悬浮颗粒。 </li><li>以10,000 xg离心1分钟。 </li><li>重复步骤1.6和1.7。弃上清。 </li><li>轻轻重悬在1毫升M9培养基。细胞可直接用于显微镜成像或稀释的10 - 100倍，以确保低细胞密度为间隔拍摄成像。请注意，低细胞密度是重要的用于扩展“延时成像。的摄像室（步骤4）在实验过程中被密封。由于指数生长的细胞中，高的初始细胞浓度可以显著耗尽室内的氧气后长时间生长在凝胶垫，减少荧光蛋白的成熟，并影响细胞的生长。 </li></ol> 3。准备的琼脂糖解<ol><li>称取10〜20毫克低熔点温度琼脂糖成1.5毫升微量离心管中。 </li><li>添加适当的音量M9基本培养基，无抗生素，3％的琼脂糖溶液。 </li><li>琼脂糖溶液加热30分钟，在70℃以熔化，反相的管，以确保该溶液是完全熔化和均匀。凝胶垫可倾在这一点上（步骤4）中，或可以将温度降低至50℃，并保持几个小时以供以后使用。 </li></ol> 4。准备在会议厅凝胶垫<ol><li>约50分钟在成像之前，漂洗Microaqueduct的的幻灯片水。 </li><li>含漱1清洗玻璃盖（使用严格的清洗方法所描述的，如在6），并通过用压缩空气吹Microaqueduct用无菌水和干滑动。 </li><li>放置的橡胶垫圈，以便它涵盖上Microaqueduct幻灯片的滑动的Microaqueduct（玻璃侧的入口和出口上，这可以应用在修改通过媒体灌注样品）。应用50μl琼脂糖溶液（步骤3）的中心的Microaqueduct幻灯片。 </li><li>顶部的琼脂糖溶液清洁，干燥的玻璃盖。 </li><li>让琼脂糖溶液在室温下放置30分钟。 </li><li>同时，制备细胞样品（步骤2）。 </li><li>小心地剥离从凝胶垫的顶部的盖玻璃。加入1.0微升的洗涤过的细胞培养物的凝胶垫的顶部。等待〜2分钟，被吸收的凝胶垫的文化。重要的是不要等了这么久，的凝胶垫overdries，但足够长的细胞被正确地坚持以硅胶垫。理想的等待时间会随温度和湿度。 </li><li>在等待过程中，干其他预清洗玻璃盖。 </li><li>量的样品，用新的玻璃盖，确保的盖玻璃和Microaqueduct的滑动以及对齐。 </li><li>组装的温度控制的生长室中，按照生产商的说明。现在它可以被用于显微镜（步骤5）的成像。 </li></ol> 5。间隔拍摄电影的成像和收购<ol><li>打开后，制造商的说明（ 例如，激光可能需要加热的〜0.5小时，在实验前）上的激光和显微镜。 </li><li>锁定组装室，舞台上插入的显微镜。生长室的温度设置在37℃下或其他所需的生长温度和物镜加热器。 </li><li>如果在室温以上的成像，焦点或凝胶垫通常会漂移显著〜15分钟后，初始温度换档。在实践中，利用这段时间找到地区的利益和修改成像脚本，作为必要的实验。 </li><li>查找在显微镜上的细胞，并使其居中一个预定义的和对准的摄像区域内存储的位置后，每一个电池单体。多于一个小区可以被成像在每个图像中采集时间窗口。为间隔拍摄成像以上米任何世代，确保成像的细胞最初来自其他细胞分离由至少几百μm，以便其他菌落在生长过程中，将不进入摄像区域。 </li><li>调整激光激发强度，因此，几乎所有的荧光分子光漂白剂在6曝光（ 图5）。 </li><li>使用自动成像脚本/日记，获得延时“的数据，使用在图3中的工作流程中的实验中描述的算法。 </li></ol>

<ol>
	<li>Hensel, Z., Feng, H. D., Han, B., Hatem, C., Wang, J., Xiao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+expression+dynamics+of+transcription+factor+revealed+by+single-molecule+noise+analysis.">Stochastic expression dynamics of transcription factor revealed by single-molecule noise analysis.</a> Nat. Struct. Mol. Biol. 19, 797-802 (2012).</li><li>Yu, J., Xiao, J., Ren, X., Lao, K., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+Gene+Expression+in+Live+Cells,+One+Protein+Molecule+at+a+Time.">Probing Gene Expression in Live Cells, One Protein Molecule at a Time.</a> Science. 311, 1600-1603 (2006).</li><li>Datsenko, K. A., Wanner, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+inactivation+of+chromosomal+genes+in+Escherichia+coli+K-12+using+PCR+products.">One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.</a> Proc. Nat. Acad. Sci. U.S.A. 97, 6640-6645 (2000).</li><li>Tobias, J. W., Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+Functional+Analysis+of+the+Ubiquitin-specific+Protease+Gene+UBP1+of+Saccharomyces+cerevisiae.">Cloning and Functional Analysis of the Ubiquitin-specific Protease Gene UBP1 of Saccharomyces cerevisiae.</a> J. Biol. Chem. 266, 12021-12028 (1991).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A Laboratory Manual. , A2.2 (2001).</li><li>Xiao, J., Elf, J., Li, G., Yu, J., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+gene+expression+in+living+cells+at+the+single-molecule+level.">Imaging gene expression in living cells at the single-molecule level.</a> Single Molecules: a laboratory manual. , 149-169 (2007).</li><li>Nagai, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+variant+of+yellow+fluorescent+protein+with+fast+and+efficient+maturation+for+cell-biological+applications.">A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.</a> Nat. Biotechnol. 20, 87-90 (2002).</li><li>Stagaman, G., Forsyth, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bright-field+microscopy+of+semitransparent+objects.">Bright-field microscopy of semitransparent objects.</a> J. Opt. Soc. Am. A. 5, 648-659 (1988).</li><li>Tobias, J. W., Shrader, T. E., Rocap, G., Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+N-end+rule+in+bacteria.">The N-end rule in bacteria.</a> Science. 254, 1374-137 (1991).</li></ol>

我们有没有公开。

CoTrAC方法可以推广到测量生产的常规的N-或C-末端的荧光蛋白融合的其它蛋白质，可能会破坏蛋白质的活性。 CoTrAC战略有三个独特的优势，目前的方法。首先，合作翻译融合确保为每个感兴趣的蛋白质分子的，一个分子中的荧光报道产生，允许实时蛋白质生产的准确计数。其次，膜 - 定位记者TSR-金星使荧光记者的单分子检测，从而能够检测在非常低的水平表达的蛋白质。第三和最重要的是，中的...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>琼脂糖（低熔融温度） </td><td>龙沙</td><td> 50100 </td><td></td></tr><tr><td> Milli-Q超纯水H 2 O </td><td></td><td></td><td></td></tr><tr><td> 5×M9盐</td><td></td><td></td><td>以下配方9 </td></tr><tr><td> 20％葡萄糖</td><td></td><td></td><td></td></tr><tr><td> 硫酸镁 </td><td></td><td></td><td></td></tr><tr><td> 氯化钙 </td><td></td><td></td><td></td></tr><tr><td> 50×MEM氨基酸溶液</td><td> Invitrogen公司</td><td> 11130-051 </td><td></td></tr><tr><td>温度控制生长房间 舞台适配器</td><td> Bioptechs </td><td> FCS-2 </td><td></td></tr><tr><td>物镜加热器</td><td> Bioptechs </td><td>模型依赖于显微镜的物镜</td><td></td></tr><tr><td> Microaqueduct幻灯片</td><td> Bioptechs </td><td> 130119-5 </td><td></td></tr><tr><td>微盖玻片</td><td> VWR </td><td> 40CIR-1 </td><td>可能是困难的源，也可从Bioptechs </td></tr><tr><td>封面玻璃/滑动垫片</td><td> Bioptechs </td><td> FCS20.75毫米</td><td></td></tr><tr><td>荧光显微镜</td><td>各个</td><td>设置示例：相干伊诺308C氩离子激光，奥林巴斯IX-81显微镜，奥林巴斯PlanApo 100X NA 1.45目标，Metamorph软件</td><td>必须有激光激发下，自动化XYZ阶段，脚本和自动对焦成像的自动化软件，能够，能够resolvin光学g单荧光蛋白</td></tr><tr><td> EM-CCD相机</td><td>各个</td><td>设置示例：安道尔IXON DU-898 </td><td>必须具有足够低噪声检测到上述背景下的单一荧光蛋白</td></tr></tbody></table>

single-molecule imaging of gene regulation in vivo using cotranslational activation by cleavage (cotrac)

我们描述的荧光显微镜法，共翻译激活由裂解（CoTrAC）图像与单分子的精确度不扰动的蛋白质的功能的情况下，在活细胞中的蛋白质分子的生产。此方法使得能够顺序，五分钟的时间窗期间在一个小区中产生的蛋白质分子的数目进行计数。它需要一个与激光激发功率密度为约0.5至1千瓦/厘米2，这是足够敏感的检测单一的荧光蛋白在活细胞中的分子的荧光显微镜。在此方法中所使用的荧光报道，由三部分组成：膜击靶序列，一个快速成熟的，黄色荧光蛋白和蛋白酶识别序列。记者感兴趣的蛋白质的N-末端的翻译融合。细胞生长在一个温度控制的显微镜载物台上。每五分钟后，细胞内的荧光分子成像（以及后来的合作unted通过分析荧光图像），并随后photobleached让只有新翻译的蛋白质在接下来的测量计数。 ，此方法产生的荧光图像，可以通过检测在每个图像中，将它们分配到单个细胞的荧光斑点，然后分配的细胞谱系的细胞进行分析。的数目计算出一个时间窗口内的一个给定的细胞产生的蛋白质的是，集成的斑点的荧光强度除以由单个荧光分子的平均强度。我们用这种方法测量的0-45的分子在单5分钟时间窗的范围中的表达水平。这种方法使我们能够测量噪声的λ阻遏CI的表达，并在系统生物学中有许多其他潜在的应用。

从CoTrAC跟踪生产的λ阻遏CI实验的典型结果示于图4和图5。在这个实验中，12个菌落成像以5分钟的间隔。在每个时间点，菌落第一自动对焦和集中的摄像区域内。接下来，中心/中心的位置，存储和一个亮场图像被收购。然后阶段易位〜0.5微米，从明场焦平面沿z轴移动的平面等分的细胞（无相位对比或微分干涉相差（DIC）的光学，半透明的对象可以被成像在稍外的聚焦平面<...

Watch this Scientific Journal Video about Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC at JoVE.com

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

我们描述的荧光显微镜法，共翻译激活由裂解（CoTrAC），对图像的生产与单分子的精确度不扰动的蛋白质的功能的情况下，在活细胞中的蛋白质分子。此方法已被用于按照随机表达动力学的一种转录因子，λ阻遏CI<sup&gt; 1</sup&gt;。

单分子成像的基因调控<em在体内</em使用转位激活裂解（CoTrAC）

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

single-molecule-imaging-gene-regulation-vivo-using-cotranslational

Research

JoVE Journal

Biology

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

9.8K 次观看.  Johns Hopkins University School of Medicine. 我们描述的荧光显微镜法，共翻译激活由裂解（CoTrAC），对图像的生产与单分子的精确度不扰动的蛋白质的功能的情况下，在活细胞中的蛋白质分子。此方法已被用于按照随机表达动力学的一种转录因子，λ阻遏CI 1。

视频: Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

单分子成像核运输

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

科研

生物学

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

激活细胞凋亡的细胞色素的细胞质显微注射 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Demonstration of Proteolytic Activation of the Epithelial Sodium Channel (ENaC) by Combining Current Measurements with Detection of Cleavage Fragments

The described methods can be used to investigate the effect of proteases on ion channels, receptors, and other plasma membrane proteins heterologously expressed in Xenopus laevis oocytes. In combination with site-directed mutagenesis, this approach provides a powerful tool to identify functionally relevant cleavage sites. Proteolytic activation is a characteristic feature of the amiloride-sensitive epithelial sodium channel (ENaC). The final activating step involves cleavage of the channel&#8217;s &#947;-subunit in a critical region potentially targeted by several proteases including chymotrypsin and plasmin. To determine the stimulatory effect of these serine proteases on ENaC, the amiloride-sensitive whole-cell current (&#916;Iami) was measured twice in the same oocyte before and after exposure to the protease using the two-electrode voltage-clamp technique. In parallel to the electrophysiological experiments, a biotinylation approach was used to monitor the appearance of &#947;ENaC cleavage fragments at the cell surface. Using the methods described, it was demonstrated that the time course of proteolytic activation of ENaC-mediated whole-cell currents correlates with the appearance of a &#947;ENaC cleavage product at the cell surface. These results suggest a causal link between channel cleavage and channel activation. Moreover, they confirm the concept that a cleavage event in &#947;ENaC is required as a final step in proteolytic channel activation. The methods described here may well be applicable to address similar questions for other types of ion channels or membrane proteins.

Demonstration of Proteolytic Activation of the Epithelial Sodium Channel ENaC by Combining Current Measurements with Detection of Cleavage Fragments

Proteolytic activation of the epithelial sodium channel (ENaC) heterologously expressed in Xenopus laevis oocytes can be demonstrated by combining current measurements with a biotinylation approach to investigate the appearance of ion channel cleavage products at the cell surface. Functionally important cleavage sites can be identified by using site-directed mutagenesis.

上皮钠通道（ENaC）的蛋白水解激活通过结合电流测量与检测裂解片段示范

The described methods can be used to investigate the effect of proteases on ion channels, receptors, and other plasma membrane proteins heterologously ...

Studying DNA Looping by Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.

研究DNA环接了单分子荧光共振能量转移

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.

A highly parallel method for measuring the site-specific cleavage of DNA at the single molecule level is described. This protocol demonstrates the technique using the restriction endonuclease NdeI. The method can easily be modified to study any process that results in site-specific DNA cleavage.

平行高通量单分子动力学测定，用于现场特异性DNA裂解

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...