作者想感谢一个Viksø尼尔森（诺维信公司）的纤维素酶的礼物。这项工作是由欧盟第七框架计划重建和BB/G016178和BB/G016194 BBSRC项目资助。

1。样品的制备我们正常的工作，无论从草本或木本植物的茎。草本材料的情况下，我们种植的植物成熟，种子开发后，我们收集干茎衰老是一旦完成。茎切成4毫米段，并放置在三个磨球2 mL样品瓶。在木质材料的情况下，样品地面粗木屑，当然木材文件，然后放置在球磨机进行进一步的处理。 样品被放置在一个机架内研磨/格式化站（图1）。这个站的顺序研磨，混合，和重量每一个样本。每个样品需要重复的数量是建立在一系列一定粒径的内在变化是确定的初步实验测试的地面材料。 2 mL样品瓶底部刺穿和材料免除小瓶选定的振动。振动臂粉准确配药允许平衡反馈控制。机器人分配4毫克/在一个标准的分析，并在大多数的情况下需要4重复。这使得20个植物样品/板进行分析。 2。预处理一旦样品被格式化，处理96孔板自动化液体处理系统（图2）。这里是一个温和的预处理植物材料加入350μL酸性或碱性溶液和适应块板加热。为了避免在分析过程中的蒸发，96孔板密封用硅胶马特。根据所研究的材料预处理的温度和时间可以进行修改。然而，最高温度100 ° C。替代程序是一个用于测试更严厉的预处理预处理脱线的材料和消除的过程pretreatmet。 3。水解<ol><li>材料预处理后，机器人会自动删除的预处理解决方案，并清洗，所有的水井，850μL25毫米醋酸缓冲液的5倍。进行冲洗是加入缓冲液的预处理解决方案，并随后删除后，让样品中的固体解决的总体积的50％。愿望的高度设置，以避免从井底吸固体液体质量的一半;有使用这种方法的固体的损失可以忽略不计。这些洗涤后在井的pH值是4.5和材料准备酶解。 </li><li>添加一种酶的混合物中含有的纤维素和半纤维素酶的解决方案是由机器人。每孔850μL酶的混合物配发板是在50 ° C摇床提出。执行标准水解为8小时，但此时可适应实验的目的。草使用的标准酶量是7 FPU / g的材料。 </li></ol> 4。还原糖的检测减少水解后释放的糖的测定是使用修改后的3 -甲基-2 -苯并噻唑啉hydrozone（MTBH）方法3。 MBTH被选定为最合适的方法，因为它是最简单的自动化和最不容易受到干扰的化合物，如蛋白质的。我们修改了机器人平台上使用这一高度敏感的方法，使其能够准确量化生物质水解液中的浓度的糖，与终体积为250μL，这是一个标准的光学96孔板。自动执行下面的所有步骤和三个独立的还原糖测定每个糖化反应。 <ol><li> 30μL蛋白水解物是从深孔板和25毫米的钠加防护罩的PCR 96孔板中的醋酸缓冲液45μL混合。 </li><li>分别加入25μL1N氢氧化钠和50μL含有0.43毫克/毫升MBTH和0.14毫克/毫升的数码地面电视解决方案。 </li><li>混合后，PCR板加热60 ° C为20分钟在热循环或类似设备提供完全相同数量的热量，所有96个井。 </li><li>被转移到一个光板所产生的反应。 </li><li>加入100μL氧化试剂（0.2％FeNH4（SO4）2，氨基磺酸0.2％和0.1％的盐酸）。拌匀，离开发展，至少1小时。 </li><li> 每个板块还应该包含50 nmol，加入100 nmol，150 nmol葡萄糖的标准反应。在620 nm处读取光板。 </li></ol> 5。代表性的成果: 分析不同类型的例子是在图3和4。结果全部获得通过使用自动化的平台。图3重呈现减少8小时，从地面杨树孵化释放糖与纤维素酶的数量不断增加的增加。图4给出了杨树样品的酸和碱预处理的影响。的NaOH预处理是更有效的促进水解过程中释放的糖的稀释硫酸预处理。水解后测糖量减少时使用大于1M的氢氧化钠浓度进行预处理。 <img src="/files/ftp_upload/3240/3240fig1.jpg" alt="图1" /> 图1。磨和96生物质能以及格式的机器人站<img src="/files/ftp_upload/3240/3240fig2.jpg" alt="图2" /> 图2液处理站的高通量糖化分析。 <img src="/files/ftp_upload/3240/3240fig3.jpg" alt="图3" /> 对不同的酶负荷释放减少杨树样本的糖等值图 3。 <img src="/files/ftp_upload/3240/3240fig4.jpg" alt="图4" /> 图4。A.对杨树样本的糖化，不同的预处理。pretreament与不同比例的硫酸在90℃30分钟后发表的糖。B.氢氧化钠预处理后糖酸预处理比在相同的温度和时间公布。

<ol>
	<li>Carpita, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+Biogenesis+of+the+Cell+Walls+of+Grasses.">Structure and Biogenesis of the Cell Walls of Grasses.</a> Annu Rev Plant Physiol Plant Mol Biol. 47, 445-476 (1996).</li><li>Gomez, L. D., Steele-King, C. G., McQueen-Mason, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustainable+liquid+biofuels+from+biomass:+the+writing's+on+the+walls.">Sustainable liquid biofuels from biomass: the writing's on the walls.</a> New Phytol. 178, 473-485 (2008).</li><li>Anthon, G. E., Barrett, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+reducing+sugars+with+3-methyl-2-benzothiazolinonehydrazone.">Determination of reducing sugars with 3-methyl-2-benzothiazolinonehydrazone.</a> Anal Biochem. 305, 287-289 (2002).</li><li>Gomez, L. D., Whitehead, C., Barakate, A., Halpin, C., McQueen-Mason, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+saccharification+assay+for+determination+of+digestibility+in+plant+materials.">Automated saccharification assay for determination of digestibility in plant materials.</a> Biotechnol Biofuels. 3, 23-23 (2010).</li></ol>

Variations of the standard saccharification protocol can be used in the same platform for determining the activity of cellulolytic enzymes (i.e. by variation of the enzyme concentrations used in a plate as well as using paper as substrate); comparison of the efficiency of several enzyme mixtures on a specific material; time course for saccharification; etc.
A standard saccharification protocol to compare the saccharification potential in different plant materials involves an eight hour hydrolysis (Figures 3 and 4). Most of the plant materials analysed requires four replicates. Under these conditions, the platform can process 80 samples/day. This analysis is being used to screen large populations of barley, maize, and brachypodium in order to establish variability in saccharification potential and the genes involved in its determination4.

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td>研磨及称重机器人</td><td> Labman自动化</td><td></td></tr><tr><td> 2毫升微管与帽</td><td> Sarstedt有限公司</td><td> 72.694 </td></tr><tr><td> 5毫米的不锈钢珠</td><td> Qiagen公司有限公司</td><td> 69989 </td></tr><tr><td> 1.2毫升Abgene平方米以及存储板</td><td>尔科技有限公司</td><td> TUL - 050 - 050C </td></tr><tr><td> 96平方米孔板的Whatman第垫</td><td>尔科技有限公司</td><td> 7704-0104 </td></tr><tr><td>液体安汉陵机器人</td><td> TECAN集团有限公司</td><td>自由埃沃200 </td></tr><tr><td>硫酸</td><td>尔科技有限公司</td><td> S/9231/PB17 </td></tr><tr><td>氢氧化钠</td><td>尔科技有限公司</td><td> BPE359 - 500 </td></tr><tr><td>醋酸钠</td><td> Sigma - Aldrich公司</td><td> S8750 - 500G </td></tr><tr><td>乙酸</td><td>尔科技有限公司</td><td> A/0420/PB17 </td></tr><tr><td> Novozyme 188 </td><td> Novozyme </td><td> DCN00214 </td></tr><tr><td> Celluclast 1.5L </td><td> Novozyme </td><td> CCN03122 </td></tr><tr><td> 96 PCR充分裙子板</td><td> Sarstedt有限公司</td><td> 72.1980.202 </td></tr><tr><td> D -葡萄糖</td><td>尔科技有限公司</td><td> G/0450/53 </td></tr><tr><td> 3 - 甲基-2 - 苯并噻唑啉酮腙盐酸盐水合物</td><td> Sigma - Aldrich公司</td><td> 129739 - 25G </td></tr><tr><td> DL -二硫苏糖醇</td><td> Sigma - Aldrich公司</td><td> D9163 - 1G </td></tr><tr><td>康宁微孔板96孔平底</td><td>尔科技有限公司</td><td> TKT - 521 - 050H </td></tr><tr><td>铵铁（III）硫酸十二水合</td><td> Sigma - Aldrich公司</td><td> F1668 - 250G </td></tr><tr><td>氨基磺酸</td><td> Sigma - Aldrich公司</td><td> 242772 - 500G </td></tr><tr><td>盐酸</td><td>尔科技有限公司</td><td> 12462-0026 </td></tr></tbody></table>

high-throughput saccharification assay for lignocellulosic materials

多糖，使植物木质纤维素生物质可以分解，产生的范围，随后可用于建立生物炼制的糖。这些原料将构成一个新的产业平台，这是可持续发展和碳中性，以取代目前对化石燃料的的依赖。在木质纤维素材料中观察到的解构顽抗是由植物细胞壁的内在属性。结晶纤维素嵌入xylans和arabinoxylans矩阵如多糖，整个结构是由木质素酚醛树脂聚合物，这也是难以消化1包裹。为了提高我们需要发现为细胞壁的糖化主要瓶颈，也屏幕突变和繁殖种群，以评估糖化2变异的植物原料的消化率。这些任务需要高吞吐量的方法，在这里，我们提出了一个分析平台，可以在96孔板格式执行糖化分析。这个平台已经发展到允许筛选不同植物物种的大量人口的纤维素消化率。我们温柔的预处理，部分酶解和糖的决心反应体积缩小，使大批自动化系统迅速进行评估。 这种自动化平台毫克的生物量，在受控条件下进行球磨，以减少植物材料，在一个可重复的方式进行了标准化的粒径。一旦样品的地面，自动格式化机器人分配到相应的水井深96孔板（图1）指定的材料和记录的金额。通常情况下，我们免除进入4口井有4分析重复相同的材料。一旦板块与植物所需的布局材料填充，他们是手动移动到液体处理站（图2）。在此站的样品受到与任稀酸或碱性温和的预处理，在孵育温度高达90 ° C随后被删除的预处理解决方案，并退回给适宜的pH为水解的样品用缓冲液冲洗。样本，然后培养的时间变长酶的混合物在50 ° C。一个等分是从水解还原糖自动MBTH比色法确定。

Watch this Scientific Journal Video about High-throughput Saccharification Assay for Lignocellulosic Materials at JoVE.com

High-throughput Saccharification Assay for Lignocellulosic Materials

描述一个简单，快速的方法确定了大量的植物生物量的样品糖化潜力。这种分析的自动化平台，涉及植物生物量在96孔板和随后的表现预处理，水解和释放糖的定量分析的准备。

木质纤维素材料的高通量检测糖化

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

high-throughput-saccharification-assay-for-lignocellulosic-materials

Research

JoVE Journal

Biology

13.2K 次观看.  University of York. 描述一个简单，快速的方法确定了大量的植物生物量的样品糖化潜力。这种分析的自动化平台，涉及植物生物量在96孔板和随后的表现预处理，水解和释放糖的定量分析的准备。

视频: High-throughput Saccharification Assay for Lignocellulosic Materials

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

使用高通量形状的RNA二级结构预测

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

科研

生物学

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 ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

高通量MHC II类分子结合分析抗原表位肽的定量分析

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

萤光素酶表达的重组病毒的高通量滴定

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

恢复成年斑马鱼的心脏的高通量应用

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

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 ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

检测miRNA靶在高通量的使用3&#39;LIFE分析

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

qPCRTag分析 - 高通量，实时定量PCR检测方法的基因分型Sc2.0

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

高通量<em&gt;斑马鱼</em&gt;能量消耗分析

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.