这项研究是由院内研究计划的NIDCR支持。

1。原理概述： 本视频演示了在执行嘴唇检测所涉及的步骤。 Cos1重组海肾荧光素酶（中国人民大学）抗原的融合，和原油中提取的细胞抗原的表达，得到了和没有净化使用。唇部检测发起的孵化与病人血清中微量井CRUD Ë中国人民大学抗原提取物。抗原抗体的混合物，然后转移到一个96孔过滤板，含蛋白质A / G的珠捕获IgG分子。洗涤后过滤板，含有蛋白质的抗体必然中国人民大学抗原是腔肠基板和光单位除了测量A / G的珠片，用光度计测量。 第1部分：生产海肾抗原的融合蛋白的质粒转染 设置：Cos1细胞在DMEM培养液，10％，使用标准的组织培养协议的FCS培养。 海肾荧光素酶融合质粒已被描述以前1。这些质粒DNA准备使用自Qiagen一个Midiprep套件。产量应约为1 -3毫克。测量DNA浓度为1000微克/毫升于-20 ° C原液和存储程序： <ol><li>前一天的转染分割COS - 1细胞到新的100 × 20毫米菜约2 × 10％在37板孵育 6 ° C. </li><li>翌日，COS - 1细胞应该是80-95％汇合。标签1.5 mL聚丙烯离心管每个质粒DNA转染。允许FuGENE 6转染试剂，这是保存在4 ° C，预热到室温。 </li><li>每个离心管加入94μLOPTI - MEM媒体。接下来添加6 FuGENE 6μLOPTI - MEM媒体而不触及池壁。 </li><li>孵育的混合物在室温下5分钟。 </li><li>添加1-2微克海肾荧光素酶的抗原融合质粒（1mg/ml的DNA的股票）构造。混合，然后在室温下15分钟的孵育混合物。 </li><li>细胞的DNA，FuGENE 6 OPTI - MEM解决方案，它均匀地滴落到Cos1细胞媒体。 </li></ol>第2部分：收获海肾抗原的融合<ol><li>两天后转染COS - 1细胞的收获。这是消除媒体和6毫升的PBS漂洗细胞发起。调迁的PBS后，移液器远离组织培养皿中的任何残留的PBS。 </li><li>加入1.4毫升冷裂解液组成，pH值7.5的50 mM的Tris，100 mM氯化钠， 氯化镁 2 5毫米，1％的Triton X - 100的，50％的甘油和蛋白酶抑制剂（2片，每50毫升的完整miniprotease抑制剂的鸡尾酒裂解缓冲液）。收获细胞与细胞刮板迅速转移裂解液的一半，每两个1.5 ml离心管放在冰上。 </li><li>一个布兰森Sonifier 150是用来打破的细胞。放置的离心管中含有冰和脉冲的细胞裂解液为5秒，5秒和5秒，2，2和4的超声设置，分别。 </li><li>两个4分钟的转速为4 ° C离心细胞裂解液在12500转后的第一个旋转，轻轻反转管，管的侧壁去除松散附着的杂物。第二自旋后，小心将上清转移，不中断的颗粒，从两管到一个新的离心管放在冰上。 </li><li>计算每μL的细胞裂解光单位（LU）的。要衡量的LU，1μL裂解液稀释8的PBS液中的一个新的离心管。直接加入100μl1X腔肠基板的稀释后的混合物，并立即测量在使用一个5秒读管一管光度计（20/20 ñ特纳科学）的发光。 </li><li>商店中国人民大学在-20 ° C -抗原裂解1-2天或不再分装的时间内存储在-80 ° C。 </li></ol>第3部分：准备一个色拉寺主板块<ol><li>首次加入450缓冲液（，pH值7.5的50 mM的Tris，100 mM氯化钠， 氯化镁 2 5毫米，1％TRITON X - 100）每一个深96井的聚丙烯微孔板以及一个血清主板。染料，酚红，在这一步，也可以包括在缓冲区中（终浓度为0.2微克/缓冲液的）作为示踪剂监测未来的血清样品除了和其他步骤的嘴唇检测。 </li><li>从每个样品中添加50μL血清中含有450μL缓冲液A的不同井注意这是一个在缓冲液A血清1:10稀释血清通常是不会被添加到主盘的最后两井，因为这是预留的缓冲空白。 </li><li>在使用主盘之前，它被广泛地动摇（1-2小时）一个旋转平台上。在主盘的血清是STABLE至少1个月在4 ° C，如果正确存放，以防止水分蒸发（或更长）。 </li><li>正如下文所述，此主控板提供10μL稀释后的血清中被反复删除针对多种抗原的血清分析。较大和较小的主控板，也可聘用。它不被使用时，用封口膜板密封。 </li></ol>第4部分：二合一检测<ol><li>聚丙烯96浅孔板用于测试血清。在第一步中，添加40缓冲液A液，每孔96使用一个8通道的微管板。 </li><li>下一步采取10μL稀释血清（1μL血清当量），从主盘，并直接将它添加到每一个工作板使用一个8通道的微管以及。 </li><li> 1 × 10 7光单位（LU）的是，每孔50缓冲液中添加了一个含中国人民大学抗原提取，是一个主混合未来制订。较低的输入（低至1 × 10 6），也可以使用，但在一个较低的动态范围的结果。使这个主混合物和中国人民大学的抗原混合物加入50μL，每孔。 </li><li>孵育在室温下1小时旋转振动筛板。 </li><li>在孵化期间，到每一个新的96孔过滤高温超导板（Millipore公司，贝德福德，马萨诸塞州）以及的底部添加5μLULTRALINK蛋白A / G的珠（皮尔斯生物技术，罗克福德，IL）在PBS的30％悬浮液。 </li><li> 1小时的潜伏期后，中国人民大学100μL抗原抗体反应混合物转移至96孔过滤高温超导板块包含A / G的珠使用一个8通道的微管蛋白。 </li><li>孵育96孔过滤板在室温下的额外1小时旋转振动筛。 </li><li>接下来，洗真空歧管上的过滤板。每孔100μL缓冲液A洗了8次，100μL的PBS的2倍。这可以执行手动或使用一个机器人移液工作站。 </li><li>最后一次洗涤后，真空是关闭的。取出滤板和印迹干燥使用一摞纸毛巾或滤纸，一定要去掉水分的顶部和底部的钢板。 </li></ol>第5部分：测量发光与数据分析设置： 一个贝特霍尔德LB 960炫酷微孔板光度计是用于确定在每口井用一个单一的喷油器的发光。一旦机，H 2 O，用蒸馏水冲洗进样器使用注射器冲洗周期。腔肠基板准备使用Promega的海肾荧光底物试剂盒制造商。通常6毫升1X腔肠基板组合（即60μL腔肠股票1​​X缓冲液加6毫升）准备启动机器，并运行一个完整的96孔板。贝特霍尔德LB 960炫酷微孔板发光分析板之前，催芽1X腔肠基板。打开一个程序文件，其中包含注入基板和阅读板的设置。对于这些测试，注入50μL腔肠基板，板发光读5秒，2秒动摇。 <ol><li>虽然板光度计有能力阅读整个板块，也可以被选中的板块的部分读（读菜单下）。启动程序，启动的板块中读取。 </li><li>运行后，酶标滤板及时删除，以防止溢出在光度计。导出成Excel格式进行分析MikroWin程序产生的数据。 </li><li>我们建议从两个独立运行的血清评估。然后，数据平均每个样品产生的LU滴度值。这些值可以进一步调整，减去缓冲区的空白。 </li><li>额外的数据分析，如确定可以取均值加3或5的控制值的标准偏差计算截止。 </li></ol>

<ol>
	<li>Burbelo, P. D., Goldman, R., Mattson, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+immunoprecipitation+method+for+quantitatively+measuring+antibody+responses+in+clinical+sera+samples+by+using+mammalian-produced+Renilla+luciferase-antigen+fusion+proteins.">A simplified immunoprecipitation method for quantitatively measuring antibody responses in clinical sera samples by using mammalian-produced Renilla luciferase-antigen fusion proteins.</a> BMC Biotechnol. 5, 22-22 (2005).</li><li>Burbelo, P. D., Leahy, H. P., Iadarola, M. J., Nutman, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Four-Antigen+Mixture+for+Rapid+Assessment+of+Onchocerca+volvulus+Infection.">A Four-Antigen Mixture for Rapid Assessment of Onchocerca volvulus Infection.</a> PLoS Negl Trop Dis. 3, e438-e438 (2009).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+and+robust+luminescent+profiling+of+anti-La+and+other+autoantibodies+in+Sj+gren&#x2019;s+syndrome.">Sensitive and robust luminescent profiling of anti-La and other autoantibodies in Sj gren&#x2019;s syndrome.</a> Autoimmunity. 139, 241-245 (2009).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+antibody+quantification+and+generation+of+whole+proteome+antibody+response+profiles+using+LIPS+(luciferase+immunoprecipitation+systems).">Rapid antibody quantification and generation of whole proteome antibody response profiles using LIPS (luciferase immunoprecipitation systems).</a> Biochem Biophys Res Commun. 352, 889-895 (2007).</li><li>Burbelo, P. D., Groot, S., Dalakas, M. C., Iadarola, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+definition+profiling+of+autoantibodies+to+glutamic+acid+decarboxylases+GAD65/GAD67+in+stiff-person+syndrome.">High definition profiling of autoantibodies to glutamic acid decarboxylases GAD65/GAD67 in stiff-person syndrome.</a> Biochem Biophys Res Commun. 366, 1-7 (2008).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+luminescence+assay+for+autoantibodies+to+mammalian+cell-prepared+insulinoma-associated+protein+2.">A new luminescence assay for autoantibodies to mammalian cell-prepared insulinoma-associated protein 2.</a> Diabetes Care. 31, 1824-1826 (2008).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serological+diagnosis+of+human+herpes+simplex+virus+type+1+and+2+infections+by+luciferase+immunoprecipitation+system+assay.">Serological diagnosis of human herpes simplex virus type 1 and 2 infections by luciferase immunoprecipitation system assay.</a> Clin Vaccine Immunol. 16, 366-371 (2009).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+quantitative+serological+detection+of+anti-cytomegalovirus+(CMV)+antibodies.">Highly quantitative serological detection of anti-cytomegalovirus (CMV) antibodies.</a> Virol J. 6, 45-45 (2009).</li><li>Burbelo, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four-antigen+mixture+containing+v-cyclin+for+serological+screening+of+human+herpesvirus+8+infection.">Four-antigen mixture containing v-cyclin for serological screening of human herpesvirus 8 infection.</a> Clin Vaccine Immunol. 16, 621-627 (2009).</li><li>Ramanathan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+luciferase+immunoprecipitation+systems+assay+enhances+the+sensitivity+and+specificity+of+diagnosis+of+Strongyloides+stercoralis+infection.">A luciferase immunoprecipitation systems assay enhances the sensitivity and specificity of diagnosis of Strongyloides stercoralis infection.</a> J Infect Dis. 198, 444-451 (2008).</li></ol>

LIPS需要最小检测优化，由于其简单，通常可以用在两个星期的嘴唇在任何特定的抗原产生高质量的数据。最耗时的步骤是克隆，并产生相应的质粒表达载体，含中国人民大学抗原融合。一旦产生这些质粒，可单个或多个抗原快速测试用嘴唇上面所述。应该指出，嘴唇是相当通用的，也可以在管格式或者一个与援助的一个BIOMEK机器人工作站2或洗板机，真空过滤的过滤板酶标检测血清。这种高度可扩展的系统可为人类自身抗原或病毒 4的全蛋白质组的小组分析并行的嘴唇。这可能是许多不同的抗体生成的配置文件由LIPS将用于3-9诊断，发现9抗原，10治疗，疫苗监测过程中，并为特定的疾病状态，和广大市民的健康广泛的影响。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>HiSpeed Plasmid Midi KIt</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>12643</td>
<td>&nbsp;</td>
<tr>
<td>100 x 20 mm tissue culture dishes</td>
<td>&nbsp;</td>
<td>Fischer</td>
<td>353003</td>
<td>&nbsp;</td>
<tr>
<td>Opti-MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>31985</td>
<td>&nbsp;</td>
<tr>
<td>FuGENE 6</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>1814443 </td>
<td>&nbsp;</td>
<tr>
<td>Complete, Mini protease inhibitor cocktail</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11836153001</td>
<td>&nbsp;</td>
<tr>
<td>Polypropylene 96 DeepWell 1 ml plate</td>
<td>&nbsp;</td>
<td>Fischer</td>
<td>12-566-120 </td>
<td>Deep well plate with lid and seal with Parafilm</td>
</tr>
<tr>
<td>Polypropylene microtiter plate</td>
<td>&nbsp;</td>
<td>Fischer</td>
<td>12-566-120</td>
<td>&nbsp;</td>
<tr>
<td>HTS Filter plate</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>MSBVN1B50</td>
<td>&nbsp;</td>
<tr>
<td>Ultralink Immobilized Protein A/G</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>53133 (10ml)</td>
<td>&nbsp;</td>
<tr>
<td>Renilla Luciferase Assay System</td>
<td>&nbsp;</td>
<td>Promega </td>
<td>E2820</td>
<td>For 2000 assays</td>
</tr>
<tr>
<td>Centro microplate luminometer</td>
<td>&nbsp;</td>
<td>Berthold</td>
<td>LB 960</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

antibody profiling by luciferase immunoprecipitation systems (lips)

为全面了解和量化抗体型材自身抗原和传染性的技术到疾病的机制，可能会产生新的见解，并阐明新的标志物substratify与不同的临床特点疾病，并更好地理解发病机制。我们已经开发出一种高度的定量方法叫荧光素酶免疫沉淀系统，用于分析病人血清抗体反应与感染有关的自身抗原和病原体的抗原（LIPS）。与酶联免疫吸附，高度敏感的嘴唇很容易实施调查体液的一个普遍的格式的不同抗原的血清学反应概况，并产生抗体滴度的动态范围可达6日志<sub&gt; 10</sub对某些抗原。在这些研究中，嘴唇对抗原板，甚至一些病原体（即艾滋病毒）的整个蛋白质组病人的体液免疫应答的定量分析，通常多用ELISA测试的单一抗原信息。此外，嘴唇也消除了生产所需的高度纯化的抗原，以及劳动力密集型的标准的酶联免疫吸附测定优化所需的步骤的时间和努力。在这里，我们提供了一个详细的描述执行嘴唇容易分析单个或多个抗原抗体反应检测技术方面的协议。

Watch this Scientific Journal Video about Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS at JoVE.com

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

唇（荧光素酶免疫沉淀系统）技术方面进行了阐述。总体方法涉及表达嵌合基因编码融合海肾荧光素酶（中国人民大学）在哺乳动物细胞中的抗原。原油中国人民大学抗原的提取，然后准备，没有净化，量化抗体免疫检测。

抗体分析的荧光素酶免疫沉淀系统（LIPS）

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

antibody-profiling-by-luciferase-immunoprecipitation-systems-lips

Research

JoVE Journal

Biology

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

26.7K 次观看.  National Institute of Dental and Craniofacial Research, National Institutes of Health. 唇（荧光素酶免疫沉淀系统）技术方面进行了阐述。总体方法涉及表达嵌合基因编码融合海肾荧光素酶（中国人民大学）在哺乳动物细胞中的抗原。原油中国人民大学抗原的提取，然后准备，没有净化，量化抗体免疫检测。

视频: Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

AC Electrokinetic Phenomena Generated by Microelectrode Structures

The field of AC electrokinetics is rapidly growing due to its ability to perform dynamic fluid and particle manipulation on the micro- and nano-scale, which is essential for Lab-on-a-Chip applications. AC electrokinetic phenomena use electric fields to generate forces that act on fluids or suspended particles (including those made of dielectric or biological material) and cause them to move in astonishing ways1, 2. Within a single channel, AC electrokinetics can accomplish many essential on-chip operations such as active micro-mixing, particle separation, particle positioning and micro-pattering. A single device may accomplish several of those operations by simply adjusting operating parameters such as frequency or amplitude of the applied voltage. Suitable electric fields can be readily created by micro-electrodes integrated into microchannels. It is clear from the tremendous growth in this field that AC electrokinetics will likely have a profound effect on healthcare diagnostics3-5, environmental monitoring6 and homeland security7.

In general, there are three AC Electrokinetic phenomena (AC electroosmosis, dielectrophoresis and AC electrothermal effect) each with unique dependencies on the operating parameters. A change in these operating parameters can cause one phenomena to become dominant over another, thus changing the particle or fluid behavior. 

It is difficult to predict the behavior of particles and fluids due to the complicated physics that underlie AC electrokinetics. It is the goal of this publication to explain the physics and elucidate particle and fluid behavior. Our analysis also covers how to fabricate the electrode structures that generate them, and how to interpret a wide number of experimental observations using several popular device designs. This video article will help scientists and engineers understand these phenomena and may encourage them to start using AC Electrokinetics in their research.

Manipulating fluids and suspended particles in the micro- and nano-scale is becoming more of a reality as enabling technologies, like AC electrokinetics, continue to develop. Here, we discuss the physics behind AC electrokinetics, how to fabricate these devices and how to interpret the experimental observations.

微电极结构产生的交流电动现象

The field of AC electrokinetics is rapidly growing due to its ability to perform dynamic fluid and particle manipulation on the micro- and nano-scale, ...

科研

生物学

Methylated DNA Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease 1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest 5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5'mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) 8. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA 2, 4, 9-11.

This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).

甲基化的DNA免疫沉淀

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

IP - FCM：免疫沉淀，流式细胞仪检测

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

在体外转录和Gaussia荧光素酶基因的上限由HeLa细胞转染

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

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 ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

翻译起始的分析在应力条件由多聚核糖体剖析

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

分析单个人类胚胎干细胞通过定量RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

选择性的mRNA翻译在哺乳动物细胞中的核糖体性能分析评估

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析：在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...