我们感谢美国地质调查局和佛蒙特水资源湖和研究中心提供资金。

1。磷类萃取<ol><li>准备一个0.25米的氢氧化钠（NaOH，40.00克/摩尔）+ 0.05 mol乙二胺四乙酸（EDTA，292.24克/摩尔）1L解决方案。 </li><li>添加30毫升的NaOH + EDTA溶液2克湿或干的土壤样品50 mL锥形管/。 </li><li>孵育16在室温下搅拌。 </li><li>离心机3000 XG 3分钟。 </li></ol> 2。样品提取液的pH值调整<ol><li>传输0.1毫升分装到1.5 mL微量离心管。 </li><li>添加0.017毫升冰醋酸2.5米，0.144 mL的0.4 M醋酸缓冲液醋酸钠（70.5％[CH 3 COONa，82.03克/摩尔]和29.5％的乙酸，pH值5.0），和0.739 mL去离子水。 </li><li>调整后的提取物的最终体积将1 mL和最终pH为5.0。 </li></ol> 3。酶联合溶液的制备<ol><li>准备1 L 0.1 M的醋酸钠（CH 3 COONa，82.03克/摩尔）解决方案，pH值5.0 。 </li><li>准备在醋酸钠缓冲等，最终浓度是每0.5个单位/毫升的步骤3.1准备2 × 10 mL的酸性磷酸酶的储备液。 酸性磷酸酶型我从小麦 （小麦胚芽，GP，一般在0.5 ü MG - 1固体，EC 3.1.3.2） 型酸性磷酸酶IV - S的马铃薯 （土豆，PP，一般4.6 ü MG - 1固体，EC 3.1.3.2） *活动单位的指定供应商1单位定义为磷酸盐解决每小时1微摩尔的解放，在37 ° C。 </li><li>复溶冻干从桔青霉 （真菌; NP1，一般在500 U 毫克 -1固体）核酸酶P1（EC 3.1.30.1）1毫升的去离子水，现在可以在4℃保存供日后使用。 </li><li> NP1;移液器，终浓度为2.5单位/毫升NP1在步骤3.2编写的10毫升管，轻轻混匀反相几次。 </li><li>离心30分钟，两个10毫升酶解决方案3000 XG。 </li></ol> 4。 P校准曲线和控制<ol><li>准备1升1 mM磷酸钾（K2HPO4，174.18克/摩尔）原液。 </li><li> 1毫米的磷酸钾储备液1.5 mL离心管中加入1 mL和执行700 0.5毫升系列稀释。 </li><li>弃管头，让你有7个稀释度范围从20 nmol 0.625 nmol P. </li><li>传输从每管80μL，在重复的行乙 - h的一个标准96孔板。 </li><li>添加醋酸钠缓冲液（3.1节）80μL行列第11和12，这是0 nmol P校准点。 </li><li>现在每列11和12包含8 0 nmol，20 nmol无机磷的参考样本。 </li><li>第10栏将包含以下控件： <ol><li>加入40μLPP + GP酶液+ 40μL醋酸钠缓冲液（3.1节）第10栏的前三个井。 </li><li>添加40μL聚丙烯+ GP + NP1酶液+ 40μL的醋酸钠缓冲到下一个三口井。 </li><li>添加40μL的醋酸钠溶液中含有10 nmol葡萄糖6磷酸（C 6 H 11的Na 2 O 9个 P &amp;bullet; XH 2 O，304.1克/摩尔）40μL聚丙烯+ GP酶液和最终以及在第10栏添加醋酸钠代替酶液的解决方案。 </li></ol></li></ol> 5。样品+酶孵化<ol><li>每个被测试的样本，将占据在一个标准的96孔板的1行的第9口井。 </li><li>从第2井1-9多达8行40μLpH值调整后的样本提取物分发。 </li><li>毕竟样品已派发*使用多道移液器分发PP + GP酶液1-3列，PP + GP + NP1列4-6和醋酸钠缓冲液（3.1节准备）列7-9。 *这一步必须快速进行，以确保所有的样品得到相当于孵化时间。 </li><li>盖有盖和孵育96孔板+酶的解决方案，控制在37和校准曲线刚好1小时° C。 </li></ol> 6。释放和背景无机磷的色度测量<ol><li>准备50毫升去离子水，下面的解决方案： *解决方案：0.1 M的抗坏血酸（C6H8O6，176.12克/摩尔）+ 0.5M三氯乙酸（Cl3CCOOH，163.39克/摩尔） B液：0.01 mol钼酸铵（（NH4）2MoO4，196.01克/摩尔） 溶液C：0.1M柠檬酸钠（（COONa）（CH2COONa）2 2H2O，294.10克/摩尔）+ 0.2 M钠砷酸（NaAsO2，129.91克/摩尔）+ 5％冰醋酸酸 （CH3CO2H，60.05克/摩尔）*解决一个每天必须做好准备。 </li><li>添加25μL溶液SDS 100μL溶液A，B液和50μL和20μL溶液C的所有井在96 - PL吃。迅速与多道移液器。 </li><li>覆盖板，并在室温下孵育30分钟。 </li><li>在任何可调微酶标仪850 nm处测量吸光度。 </li></ol> 7。磷化合物的分类<ol><li>原始数据导入到电子表格应用程序（如Microsoft Excel）。 </li><li>剧情无机磷校准曲线：在X轴的0至40 nmol P和Y轴重复的吸光度测量平均。 </li><li>进行线性回归，并寻找最佳拟合线方程。 </li><li>列1-3：直接适用的校准曲线，这是背景无机体育</li><li>列4-6：平均一式三份样本值，减去从总吸收，这是P水解P单酯衍生的控制（酶液+背景的无机磷）。 </li><li>列7-9：与7.5相同，但减去吸光度值列4-6 - 这是从水解P酯P。 </li><li>吸光度数据转换nmol P应用的校准方程发布。 </li><li> nmol P发布，可与毫克/公斤的原土。此外，评估每个P类的比例，也可能是一个有用的方法分析数据。 </li></ol> 8。代表性的成果： 快速目视检查后96孔板比色化学，将提供程序是否正确执行的线索。首先要检查的液体在每口井的水平扫描板的侧面。应该有整整275μL试剂在所有井。下一步，目视检查一式三份样品孔的颜色和重复校准水井。这些技术的复制应该是相同的蓝色色调。下一步申请校准曲线含葡萄糖6磷酸的两口井，并确认他们发行后总提取P所有10 nmol无机体育已测使用ICP - OES或一种替代方法，确保使用这个值来计算的总P协议不超过P的提取量。 注：认真在电子表格中的数据管理，将有助于对定量错误的后卫。一次，您将处理大量的数字，所以创建一个模板，将花费的时间。 <img src="/files/ftp_upload/2660/2660fig1.jpg" alt="图1" /> 图1显示8个样品（行AH）和校准曲线（列11和12）的结果的96孔板。控制在10列。列1-3和4-6之间的颜色强度的增加是由于水解酯P化合物，列4-6和7-9之间，是由于水解酯P化合物。 <img src="/files/ftp_upload/2660/2660fig2.jpg" alt="图2" /> 图2，一个0.25米的NaOH - 0.05 M EDTA佛蒙特州土壤样本，采用高通量酶解提取的P类分布。误差线表示标准偏差，N = 3。

<ol>
	<li>Turner, B. L., Frossard, E., Baldwin, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Organic phosphorus in the environment. , (2005).</li><li>Murphy, J., Riley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+single+solution+method+for+determination+of+phosphate+in+natural+waters.">A modified single solution method for determination of phosphate in natural waters.</a> Anal. Chim. Acta. 27, 31-36 (1962).</li><li>Dick, W. A., Tabatabai, M. A. <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+orthophosphate+in+aqueous+solutions+containing+labile+organic+and+inorganic+phosphorus+compounds.">Determination of orthophosphate in aqueous solutions containing labile organic and inorganic phosphorus compounds.</a> J. Environ. Qual. 6, 82-85 (1977).</li><li>He, Z., Toor, G. S., Honeycutt, C. W., Sims, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enzymatic+hydrolysis+approach+for+characterizing+labile+phosphorus+forms+in+dairy+manure+under+mild+assay+conditions.">An enzymatic hydrolysis approach for characterizing labile phosphorus forms in dairy manure under mild assay conditions.</a> Bioresour. Technol. 97, 1660-1668 (2006).</li><li>He, Z., Honeycutt, C. 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+modified+molybdenum+blue+method+for+orthophosphate+determination+suitable+for+investigating+enzymatic+hydrolysis+of+organic+phosphates.">A modified molybdenum blue method for orthophosphate determination suitable for investigating enzymatic hydrolysis of organic phosphates.</a> Commun. Soil Sci. Plant Anal. 36, 1373-1383 (2005).</li><li>Cade-Menun, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+phosphorus+in+environmental+and+agricultural+samples+by+31P+nuclear+magnetic+resonance+spectroscopy.">Characterizing phosphorus in environmental and agricultural samples by 31P nuclear magnetic resonance spectroscopy.</a> Talanta. 66, 359-371 (2005).</li><li>Johnson, N. R., Hill, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorus+species+composition+of+poultry+manure-amended+soil+using+high-throughput+enzymatic+hydrolysis.">Phosphorus species composition of poultry manure-amended soil using high-throughput enzymatic hydrolysis.</a> Soil Sci. Soc. Am. J. 74, 1786-1791 (2010).</li></ol>

就其性质而言，快速的方法，使用小容量，需要格外小心。因此，最重要的步骤是那些涉及到的板块吹打解决方案。准确，和最重要的，一致的吸管技术的是这个实验的成功至关重要。 的NaOH - EDTA的提取将使大部分在许多样品的p特点分为三类：磷酸盐，P单酯和双酯体育土壤，肥，沉积物或任何其他环境样品中含有氢氧化钠提取的P - EDTA可以特点。环境样品中的P形式不一定是稳定的，这种技术将确保样品的特点，样品前妥协，而无需聘用的研究人员的军队。 这个实验是特别适合时要处理大量的样品。该试剂的需求和空间的要求已经缩减到一个可控的水平（如1.5毫升离心管，而不是50 mL的玻璃烧瓶）。这种适应也限制了废物的产生。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td>氢氧化钠</td><td> Sigma - Aldrich公司</td><td> S8045 </td></tr><tr><td> EDTA </td><td> Sigma - Aldrich公司</td><td> EDS公司</td></tr><tr><td>冰醋酸</td><td> Sigma - Aldrich公司</td><td> 242853 </td></tr><tr><td>醋酸钠</td><td> Sigma - Aldrich公司</td><td> S2889 </td></tr><tr><td>小麦酸性磷酸酶</td><td> Sigma - Aldrich公司</td><td> P3627 </td></tr><tr><td>马铃薯酸性磷酸酶</td><td> Sigma - Aldrich公司</td><td> P1146 </td></tr><tr><td>核酸酶P1 </td><td> Sigma - Aldrich公司</td><td> N8630 </td></tr><tr><td>磷酸钾</td><td> Sigma - Aldrich公司</td><td> P2222 </td></tr><tr><td></td><td> Sigma - Aldrich公司</td><td></td></tr><tr><td></td><td> Sigma - Aldrich公司</td><td></td></tr><tr><td>葡萄糖6磷酸</td><td> Sigma - Aldrich公司</td><td> G7250 </td></tr><tr><td> SDS </td><td> Sigma - Aldrich公司</td><td> L4390 </td></tr><tr><td>抗坏血酸的</td><td> Sigma - Aldrich公司</td><td> A5960 </td></tr><tr><td>三氯乙酸</td><td> Sigma - Aldrich公司</td><td> T9159 </td></tr><tr><td>钼酸铵</td><td> Sigma - Aldrich公司</td><td> A1343 </td></tr><tr><td>柠檬酸钠</td><td> Sigma - Aldrich公司</td><td> S1804 </td></tr><tr><td>钠砷</td><td> Sigma - Aldrich公司</td><td> S9663 </td></tr></tbody></table>

high-throughput measurement and classification of organic p in environmental samples

许多类型的有机磷（P）的分子存在于环境样品1。传统的P的测量并没有发现这些有机磷化合物，因为它们不与 2,3比色试剂反应。酶法水解（EH）是一个新兴的方法，准确定性 4,5环境样品中的有机磷形式。这种方法只莫须有的准确性，磷- 31核磁共振光谱仪（31个P - NMR）的方法，价格昂贵，需要专门的技术training6 。我们已经适应了酶解方法，能够测量三磷类（酯磷和无机磷酯磷，），酶标仪系统 7 。这种方法提供了一种快速，准确，价格合理，用户友好的方式来衡量，在土壤，沉积物，粪肥和，如果集中，水产品样品的P物种的研究人员。这是唯一的高通量方法的测量可以在一个标准的实验室进行的形式和有机磷酶不稳。由此产生的数据提供了洞察科学家研究系统养分含量及富营养化的可能性。

Watch this Scientific Journal Video about High-Throughput Measurement and Classification of Organic P in Environmental Samples at JoVE.com

High-Throughput Measurement and Classification of Organic P in Environmental Samples

此协议，介绍了一种酶水解，利用酶标仪来衡量和分类，土壤磷P单酯，磷酯和无机体育多达96个样品，可以在一次在一个标准的实验室测量高通量的方法。

高通量测量和环境样品中的有机磷分类

Many types of organic phosphorus (P) molecules exist in environmental samples1.  Traditional P measurements do not detect these organic P compounds since ...

high-throughput-measurement-classification-organic-p-environmental

Research

JoVE Journal

Biology

12.5K 次观看.  University of Vermont. 此协议，介绍了一种酶水解，利用酶标仪来衡量和分类，土壤磷P单酯，磷酯和无机体育多达96个样品，可以在一次在一个标准的实验室测量高通量的方法。

视频: High-Throughput Measurement and Classification of Organic P in Environmental Samples

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

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

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

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

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 ...

科研

生物学

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 ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

粉末样品的绝对量子产量测量

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

化学阻断抗体芯片复用高通量的复杂样品中特定蛋白糖基化的剖析

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

高通量清醒小鼠肾小球滤过率的测定方法

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

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 ...

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 ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.
Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.
This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

流式细胞仪检测盘基柄柄Macropinocytosis 的高通量

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...

High-throughput Measurement of Gut Transit Time Using Larval Zebrafish

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and pharmacological similarities to that of the mammals. GI intolerance during clinical testing of drug candidates is common and may pose a serious threat to human health. Testing for GI toxicity in preclinical mammalian models can be expensive in terms of time, test compound, and labor. The high-throughput method presented here may be used to predict GI safety issues. Compared to mammalian models, this method allows for more expedient assessment of test compound effects on GI transit while using low quantities of compound. In this method, larval zebrafish (7 days post fertilization) are fed food containing a fluorescent label. After feeding, each larval fish is placed into a well of a 96-conical-bottom-well plate and dosed with test compound (dissolved in water) or the vehicle. As gut transit occurs, fecal matter accumulates on the bottom of the wells, and the rate at which this happens is monitored by measuring fluorescence from the bottom of the well repeatedly over time using a plate spectrophotometer. The fluorescence from larvae in a given treatment group are averaged and these values are graphed along with standard error, for each measurement time, yielding a curve representing average transit of food over time. Effects on gut transit time are identified by comparing the area under the curve for each treatment group to that of the vehicle-treated group. This method detected changes in zebrafish GI transit time induced by drugs with known clinical GI effects; it can be employed to interrogate dozens of treatments for GI effects per day. As such, safer compounds can be quickly prioritized for mammalian testing, which expedites discovery and proffers 3Rs advancement.

The goal of this protocol is to measure the transit time of fluorescently labeled food through the gut of larval zebrafish in a high throughput fashion.

利用斑马鱼对肠道转运时间进行高通量测量

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;