线虫转基因线虫遗传学中心（美国明尼苏达大学，明尼阿波利斯，明尼苏达州）。这项工作是支持由美国国立卫生研究院R21授予NS0667678 - 01到金水。参加所有作者在收集，分析和解释数据。 CKL，KS和KPC参加了编写和修改的手稿。

1。细菌的蠕虫食品的制备第1天<ol><li>加入5毫升饱和大肠杆菌大肠杆菌 OP50细菌培养到500毫升了不起的肉汤补充50微克/ ml链霉素和成长在一个摇床（225转）隔夜在37 ° C。 </li></ol>第2天<ol start="2"><li> 2500 RCF为20分钟，分割成10个50毫升管和一个冷冻离心机离心机细菌过夜的细菌培养。 </li><li>倒出关闭LB培养基，并在每10毫升的液体线虫的生长介质（NGM）重悬菌体。握在地板摇床水平为15分钟悬浮细菌。为了使NGM的缓冲区，添加3 g氯化钠至1 L去离子水和高压灭菌。冷却至55 ° C，并在顺序添加无菌解决方案如下：1毫升1个M 氯化钙 ， 硫酸镁的1 M 1毫升，25毫升1 M磷酸钾，pH值6.0。 </li><li>在冷冻离心机离心20分钟，在2500 RCF的细菌培养。 </li><li>倒出关闭NGM的缓冲区，并权衡菌体。 </li><li>添加NGM的缓冲液等体积​​的悬浮颗粒，等分3毫升细菌集中到15毫升的试管，并储存在-20 ° C。 </li></ol> 2。大规模C.线虫液体培养我们使用了转基因株系VP596（dvIs19 [pAF15（GST - 4：：GFP：免入息审查贷款计划 ）]; vsIs33 DOP - 3：RFP），携带两个 ​​荧光结构：Pgst -4：：GFP 的 14监察SKN - 1活动和PDOP - 3：RFP的 17作为一个蠕虫号码规范化标准。 第1天<ol><li>混合150毫升NGM的缓冲液，1.5毫升的LB肉汤，150μL5 mg / ml的胆固醇（乙醇），100毫克/ ml链霉素和75μL。过滤消毒的混合物，并添加1升的无菌烧瓶。 注：NGM的缓冲液中添加1％LB肉汤有助于坚持玻璃器皿和塑料制品，以防止蠕虫，但这一数额是不够的支持细菌生长。 </li><li>同步标准次氯酸钠蠕虫程序18。在一个15毫升管次氯酸钠溶液5毫升（3.75毫升无菌水，1毫升的家用漂白剂，和250μL10 N的氢氧化钠）可处理多达0.5毫升妊娠蠕虫。发布鸡蛋用无菌水清洗和重新悬浮在10毫升NGM的缓冲区。 </li><li>稀释100倍NGM的缓冲区（例如，100μL于10 ml）的鸡蛋样本，并在三个独立的5μL分装鸡蛋的数量计数。多200,000（100稀释倍数x 2000）的平均人数估计总数的鸡蛋。 </li><li>新增20万到2万只鸡蛋NGM的缓冲瓶和动摇的文化，在100转20 ° C。 </li></ol>第2天<ol start="5"><li>烧瓶中加入鸡蛋，至少有16小时后使用无菌血清吸管取出约0.5毫升悬浮蠕虫文化。吸取三个5μL下降到无菌培养皿盖子。在-20 ° C冰箱中放置1-2分钟瘫痪蠕虫的盖子。计数孵化每5μL，然后由30000（150毫升/ 5μL）多活蠕虫的平均人数估计总数孵出的蠕虫。 </li><li>解冻一管冷冻OP50细菌培养议定书“（1.6）和3毫升50％OP50添加到每50万孵出的蠕虫蠕虫文化。摇烧瓶中，在100转20 ° C。蠕虫会发展成L4幼虫，并在51小时左右的青壮年。开始时，细菌的OD600值应约为2.200。监测细菌的吸光度量和添加更多的外径600下降到低于0.900。 </li></ol> 3。蠕虫收集和配药第4天<ol><li>使用无菌血清吸管转移约0.5毫升悬浮蠕虫文化，一个标准的NGM琼脂平板。立体显微镜，以确保大多数是L4幼虫或青壮年蠕虫。 L4幼虫会在身体的中间有一个腹斑点清晰。年轻的成年人将稍大，不会有一个明确的点。 </li><li>倒入无菌50毫升管蠕虫的文化，并将其放置在试管架台式管。允许蠕虫解决约10分钟。取出吸入或移液上清。 注意：这一步也很重要，以除去未孵化的鸡蛋或蠕虫没有发展，因为他们将解决速度比L4幼虫和年轻的成年人。 </li><li>收集到一个单管50毫升和1％的LB肉汤（NGM +的LB）3-4次，以去除细菌NGM的缓冲区清洗所有的蠕虫。 注：这是最好的蠕虫为30秒500 RCF通过收集和更换新鲜NGM +的LB缓冲区上清液中摆动桶离心机。 </li><li>洗涤后，填写NGM管+的LB缓冲至50毫升，倒入一个自定义的蠕虫配药烧瓶。添加一个小搅拌棒，搅拌暂停保持蠕虫。数日E的蠕虫数量在3个5μL滴如上。 NGM +的LB稀释，直到你每5μL滴（〜2.5-3.0蠕虫/μL）12-15蠕虫。 注：约20毫升（60,000蠕虫）最低需要填补配药烧瓶和饮水机卡带的死腔。每384孔板需要约35000蠕虫或暂停蠕虫的11.5毫升。 </li><li>负载10μL配药卡带和消毒用70％乙醇的吸。吸用无菌水冲洗出来的乙醇。 </li><li>因此，它只是上述不影响其运动的情况下，搅拌棒插入到烧瓶配药卡带年底。确保蠕虫仍然在整个烧瓶暂停。计划饮水机，免除在低速2预脉冲。总理和运行至少10毫升的暂停蠕虫。填写需要迅速解决油管，以防止蠕虫的板。 </li><li>透气胶带密封板。 </li><li>放置在孵化器的平台，在适当的温度，为您的检测握手的板块。请勿堆叠板。 </li></ol> 4。荧光分析<ol><li>目标板放入酶标仪测量每口井的荧光强度与相应的发射和激发波长（我们的检测过滤器：GFP 485/20ex 528/20em; RFP的540/25ex 590/35em）。 </li><li>计算GFP / RFP的比例正常化，并与对照孔（不激活化合物）的读数从个别治疗所产生的荧光强度，以确定确切倍的差异。 </li></ol> 5。代表性的成果： 我们的SKN - 1法使用一个染色体整合双记者株（VP596）。正如图1所示，蠕虫以及相关量配药。在每口井的图2A和2B，总GFP和RFP的荧光显示的高度重复性，以及跨384孔板。如图所示，图2C，联合国诱导Pgst - 4：：GFP荧光线性关系PDOP - 3：RFP的跨384孔。当作为一个比GFP / RFP表达，荧光变得高度重复性以及跨384孔板展示PDOP - 3的能力（比较从图2D到2A的变异系数）从：RFP的记者，以减少变异。如在图3中，RFP与GFP的相对感应所示的一个SKN - 1激活异生（38微米胡桃）是强大的和高度跨384孔板的重复性。 <img src="/files/ftp_upload/2745/2745fig1.jpg" alt="图1"/> 图1。蠕虫与量数配发。蠕虫分别稀释至约2/μl和配发到24孔板与体视显微镜手动计数。 N = 8口井每卷。 <img src="/files/ftp_upload/2745/2745fig2.jpg" alt="图2"/> 图2到384孔板配发蠕虫的总荧光重现。配发到每一个384孔板以及蠕虫稀释至约2.5/μl和30μL。 GFP（一）和RFP（二）荧光所有井的变异系数低于9％。 （三）绿色荧光高度相关RFP的荧光。 （四）计算RFP的绿色荧光蛋白的比例减少的变异系数低于6％（A，B，和D）的实线表示的手段和虚线表示三个高于或低于平均值的标准偏差。 <img src="/files/ftp_upload/2745/2745fig3.jpg" alt="图3"/> 图3激活Pgst - 4：绿色荧光蛋白是强大的和一致的。配发到一个384孔板和38微米胡桃所有井约75 L4蠕虫中添加了其他所有列。 GFP和RFP的荧光测定孵化后21 h。平均相对荧光比率（1.0）的所有控制和胡桃井（8.9）都标有实线。控制上述三个标准差的均值和胡桃意味着下面的虚线标记。

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我们提出了培养方法，并配发大量的转基因线虫。培养蠕虫使用的设备是标准的实验室进行分子克隆和液体处理和设备的荧光微孔板处理大量的实验室标准。配药的活C.大量的其他方法线虫需要昂贵的粒子分拣设备19。 Pgst - 4：绿色荧光蛋白检测可用于屏幕上的第“N”白领转录因子的小分子调制器和检测异生和氧化剂污染物在环境和食品样品14,16。乐百氏诱导?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> LB培养基</td><td>研究产品国际公司</td><td> L24066 </td><td></td></tr><tr><td>太棒了肉汤</td><td>研究产品国际公司</td><td> T15100 </td><td></td></tr><tr><td>协同™HT多模酶标仪</td><td>百特</td><td></td><td>过滤器：GFP 485/20ex 528/20em; RFP的540/25ex 590/35em </td></tr><tr><td> MicroFlo选择饮水机</td><td>百特</td><td></td><td></td></tr><tr><td>蠕虫配药烧瓶</td><td>南部科学公司，佛罗里达州，Micanopy </td><td></td><td>自定义组装（352）284-2531 </td></tr><tr><td> 384微孔板</td><td>格雷纳生物的一</td><td> 5678-1209 </td><td></td></tr><tr><td>透气封箱胶带</td><td> NUNC </td><td> 241205 </td><td></td></tr><tr><td> （5 - 羟基- P - naphthoqinone）胡桃</td><td>阿库罗斯福冈</td><td> 121640010 </td><td>胡桃是溶于DMSO </td></tr><tr><td>二甲基亚砜</td><td> Sigma - Aldrich公司</td><td> D - 5879 </td><td></td></tr><tr><td> 线虫转基因株</td><td>作者实验室</td><td> VP596 </td><td> Pgst - 4：：GFP和PDOP - 3：招标书 </td></tr></tbody></table>

high-throughput screening and biosensing with fluorescent c. elegans strains

高通量筛选（HTS）是一个强大的方法确定的生物过程的化学调制。然而，在使用细胞培养模型的屏幕中发现了许多化合物， 经常 发现 在 1-2体内有毒或药物活动。在整体动物模型的筛选，可以帮助避免这些缺陷，并简化药物开发的路径。 线虫是一个多细胞生物模型，非常适合用于高通量筛选。这是小（&lt;1毫米），并可以在经济上的培养，在液体中的处方。 C。 线虫也是最听话的实验动物模型允许快速和详细的鉴定药物模式的行动之一。 我们描述了培养协议，并配发荧光的 C株线虫为高通量筛选化学库或改变特定基因的表达的环境污染物的检测。发展同步蠕虫的大量液体培养基中生长，收获，洗涤，并悬浮在一个定义的密度。蠕虫，然后添加到黑，平底的384孔板使用蠕动的液体分配器。从一种化学品库或测试样本（例如，水，食物，或土壤）的小分子可以被添加到井与蠕虫体内，实时荧光强度与荧光酶标仪测定。这种方法可以适应任何C.诱导基因一个合适的记者提供的线虫。许多诱导应力和发展的转录途径是定义在 C 线虫和GFP的转基因记者菌株已经存在许多其中4人。当与适当的转基因记者结合，我们的方法可用于屏幕通路调制器，或发展为强大的生物传感器检测环境污染物。 我们证明我们的C 。 线虫文化和配药协议，高温超导实验，我们发展到显示器的 C. 线虫第“N”白领转录因子SKN - 1。 SKN - 1和哺乳动物同源Nrf2的激活过程中的氧化和异生强调5-10细胞保护基因。 Nrf2的保护哺乳动物和许多与年龄有关的，如癌症，神经退行性疾病，和慢性炎症疾病已成为一个主要的化疗目标11-13。我们的实验是基于GFP的转基因SKN - 1靶基因 GST -4 14记者，编码谷胱甘肽- S转移6。 商品及服务税-4记者也是一个传感器异生和氧化的化学物质，激活SKN - 1，可用于检测水平低的污染物，如丙烯酰胺和甲基水银15-16。

Watch this Scientific Journal Video about High-throughput Screening and Biosensing with Fluorescent C. elegans Strains at JoVE.com

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

液为基础的培养和分配过程<em&gt; C。线虫</em&gt;菌株表达荧光记者蛋白质的描述，不需要昂贵的分拣设备。这种方法可以适用于众多诱导<em&gt; C。线虫</em&gt;基因药物发现或污染物的生物传感。

高通量筛选和生物传感与荧光 C。线虫菌株

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

high-throughput-screening-biosensing-with-fluorescent-c-elegans

Research

JoVE Journal

Biology

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

17.8K 次观看.  University of Florida. 液为基础的培养和分配过程 C。线虫菌株表达荧光记者蛋白质的描述，不需要昂贵的分拣设备。这种方法可以适用于众多诱导 C。线虫基因药物发现或污染物的生物传感。

视频: High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

真菌内切葡聚糖酶的活性高通量筛选大肠埃希氏菌</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

科研

生物学

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 ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

定量和自动化高通量的全基因组RNAi屏幕 C。线虫</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

高通量的亲和标签的重组蛋白的纯化

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

内向整流钾通道的小分子调节剂的高通量筛选

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

线虫趋含量的

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

生化和高通量的微观评估的脂肪含量<em&gt;秀丽隐杆线虫</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

使用自制双辉光萤光素酶检测的高通量筛选功能

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

激酶底物对鉴定使用高通量筛选


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...