这项工作是由肝炎病毒研究的研究所支持。

<ol>
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作者没有透露。

1。目标蛋白质和捕获抗体的选择抗体芯片检测之前，一些试剂和材料都需要考虑和准备。聚糖分析或聚糖的生物标志物的筛选，抗体的特定糖候选人的面板设计的抗体芯片，根据文献，或从先前的结果，应确定。购买来自不同厂商，如R＆D系统等的IgG，这些抗体通常是首选，因为我们以前的测试表明，一些IgM和IgE可能完全失去了化学修饰后，其抗原的亲和力捕获抗体。 <p cla...

<table border="1"><tbody><tr><td> 编号 </td><td> 试剂名称 </td><td> 缩写 </td><td> 公司 </td><td> 产品编号 </td></tr><tr><td>母语</td><td>生物素伴刀豆球蛋白A </td><td>刀豆</td><td>媒介实验室</td><td> BK-1000 </td></tr><tr><td>二级</td><td>接骨木黑质凝集素生物素</td><td>国民核算体系</td><td>媒介实验室</td><td> B - 1305 </td></tr><tr><td>三级</td><td>生物素镜头扁豆凝集素</td><td> LCA的</td><td>媒介实验室</td><td> BK-2000 </td></tr><tr><td> L4 </td><td>生物素蓖麻凝集素我</td><td>的RCA </td><td>媒介实验室</td><td> BK-1000 </td></tr><tr><td>腰</td><td> Aleuria Aurantia凝集素生物素</td><td> AAL的</td><td>媒介实验室</td><td>的B-1395 </td></tr><tr><td> L6 </td><td>刺桐Cristagalli凝集素生物素</td><td>的ECL </td><td>媒介实验室</td><td> BK-3000 </td></tr><tr><td> L7的</td><td>生物素加纳（Bandeiraea）的Simplicifolia凝集素二</td><td>吉斯达二</td><td>媒介实验室</td><td> BK-3000 </td></tr><tr><td>八楼</td><td>生物素小麦胚芽凝集素</td><td> WGA的</td><td>媒介实验室</td><td> BK-1000 </td></tr><tr><td>采用L9 </td><td>生物素绿豆寻常Erythroagglutinin </td><td> PHA-E的</td><td>媒介实验室</td><td> BK-2000 </td></tr><tr><td> L10 </td><td>生物素绿豆寻常Leucoagglutinin </td><td> PHA-L的</td><td>媒介实验室</td><td> BK-2000 </td></tr><tr><td> L11 </td><td>生物素标记的花生凝集素</td><td>巴勒斯坦民族权力机构</td><td>媒介实验室</td><td> BK-1000 </td></tr><tr><td> L12 </td><td>生物素豌豆凝集素</td><td>变压吸附</td><td>媒介实验室</td><td> BK-2000 </td></tr><tr><td> L13号</td><td> Dolichos Biflorus凝集素生物素</td><td> DBA的</td><td>媒介实验室</td><td> BK-1000 </td></tr><tr><td> L14 </td><td>生物素曼陀罗曼陀罗凝集素</td><td>的DSL </td><td>媒介实验室</td><td> BK-3000 </td></tr><tr><td> L15 </td><td>生物素苦参粳稻凝集素</td><td> SJA </td><td>媒介实验室</td><td> BK-2000 </td></tr><tr><td> L16号</td><td>生物素标记的大豆凝集素</td><td>校本评核</td><td>媒介实验室</td><td> BK-1000 </td></tr><tr><td> L17 </td><td>生物素化的马铃薯（土豆）凝集素</td><td> STL的</td><td>媒介实验室</td><td> BK-3000 </td></tr><tr><td> L18的</td><td>生物素加纳（带eiraea）Simplicifolia凝集素我</td><td>吉斯达我</td><td>媒介实验室</td><td> BK-2000 </td></tr><tr><td>第19课</td><td>蚕豆簇毛麦凝集素生物素</td><td> VVL </td><td>媒介实验室</td><td> BK-2000 </td></tr><tr><td> L20的</td><td>生物素化的番茄（番茄）凝集素</td><td> LEL的</td><td>媒介实验室</td><td> BK-3000 </td></tr><tr><td>为L21 </td><td>生物素标记的荆豆凝集素我</td><td> UEA的我</td><td>媒介实验室</td><td> BK-1000 </td></tr><tr><td> L22的</td><td>生物素Jacalin </td><td> JACALIN </td><td>媒介实验室</td><td> BK-3000 </td></tr><tr><td> A1的</td><td>山羊F（下AB&#39;）2片段的抗人IgM，Fc5μ抗体</td><td> IgM抗体</td><td>杰克逊免疫研究</td><td> 109-006-129 </td></tr><tr><td> A2的</td><td>驴F（AB&#39;）2破片抗人IgG（H + L）tibody </td><td> AB1类</td><td>杰克逊免疫研究</td><td> 709-006-149 </td></tr><tr><td> A3的</td><td>鼠抗人IgG F（AB&#39;）2单克隆抗体</td><td> AB3 </td><td>杰克逊免疫研究</td><td> 209-005-097 </td></tr><tr><td> A4纸</td><td>羊抗人α2巨球蛋白的多克隆抗体</td><td> A2M开发</td><td> GeneTex </td><td> GTX62924 </td></tr><tr><td> A5的</td><td>兔抗人α-1-抗胰蛋白酶多克隆抗体</td><td> A1AT </td><td>李Biosiences </td><td> CA1T-80A </td></tr><tr><td> A6的</td><td>鼠抗人α-1-抗胰蛋白酶单克隆抗体</td><td> A1AT </td><td> Sigma Aldrich公司</td><td> SAB4200198 </td></tr><tr><td> A7的</td><td>兔抗人α-1-抗胰蛋白酶多克隆抗体</td><td>法</td><td> NeoMarkers </td><td> RB-367-A1的</td></tr><tr><td> A8的</td><td>兔抗人α-1-抗糜蛋白酶多克隆抗体</td><td>法</td><td> Fisher Scientific则</td><td> RB9213R7 </td></tr><tr><td> A9的</td><td>鼠抗人转铁蛋白的单克隆抗体</td><td>转</td><td> GeneTex </td><td> GTX101035 </td></tr><tr><td> A10的</td><td>兔抗人转铁蛋白的多克隆抗体</td><td>转</td><td> GeneTex </td><td> GTX77130 </td></tr><tr><td> A11 </td><td>羊抗人载脂蛋白J多克隆抗体</td><td> ApoJ </td><td> abcam </td><td> ab7610 </td></tr><tr><td> A12往</td><td>鼠抗人GP73单克隆抗体</td><td> GP73 </td><td>雅培</td><td> 14H4-23 </td></tr><tr><td> A13号</td><td>鼠抗人GP73单克隆抗体</td><td> GP73 </td><td>圣克鲁斯生物技术公司</td><td> SC-101275 </td></tr><tr><td> A14高速公路</td><td>兔抗人α-1 fetoprotein多克隆抗体</td><td>法新社</td><td>建屋</td><td>幸福感的41C966 </td></tr><tr><td> A15 </td><td>鼠抗人的α-1单克隆抗体甲胎蛋白</td><td>法新社</td><td>菲茨杰拉德</td><td> 10-A05A </td></tr><tr><td> A16 </td><td>鼠抗人单克隆抗体hemopexin </td><td> hemopexin </td><td> assaypro </td><td> 60190-05011 </td></tr><tr><td> A17中</td><td>鼠抗人的glypican，3（1G12）单克隆抗体</td><td> GPL3 </td><td>圣克鲁斯生物</td><td> SC-65443 </td></tr><tr><td> A18 </td><td>鼠抗人激肽原（低分子量）单克隆抗体</td><td>激肽原</td><td> assaypro </td><td> 20333-05011 </td></tr><tr><td> A19中</td><td>兔抗人MMP-21单克隆抗体</td><td> MMP21 </td><td> epitomic </td><td> 1955-1 </td></tr><tr><td> A20的</td><td>鼠抗人CEACAM-1单克隆antibod的Ÿ </td><td> CEACAM </td><td> R＆D系统</td><td> MAB1180 </td></tr><tr><td> A21号</td><td>鼠抗人DPPIV/CD26单克隆抗体</td><td> DPPIV </td><td> R＆D系统</td><td> MAB22441 </td></tr><tr><td> A22号</td><td>鼠抗人PIVKA第二单克隆抗体</td><td> PIVICA </td><td>晶体化学</td><td> 8040 </td></tr><tr><td> A23座</td><td>鼠标防癌胚抗原</td><td>东航</td><td>美国生物</td><td> C1300 </td></tr><tr><td> A24的</td><td>小鼠抗血清CA125癌抗原</td><td> CA125的</td><td>美国生物</td><td> C0050-01D </td></tr><tr><td> A25 </td><td>鼠标反CA19-9癌抗原</td><td> CA19-9 </td><td>美国生物</td><td> C0075-18 </td></tr><tr><td> A26 </td><td>鼠标反刘易斯x的单克隆抗体</td><td>刘易斯X </td><td> Calbiochem公司</td><td> 434631 </td></tr><tr><td>生物</td><td>生物素标记的牛血清白蛋白（阳性对照） </td><td>生物</td><td>自制</td><td> N / A </td></tr></tbody></table> 表1。在这个协议中使用的凝集素和抗体的名单。 <table border="1"><tbody><tr><td> 试剂小号/设备名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>非接触微阵列</td><td> BioDot公司</td><td> sciFLEXARRAYER </td></tr><tr><td> 384微孔板</td><td>费舍尔</td><td> 14-230-243 </td></tr><tr><td> FoodSaver </td><td> FoodSaver </td><td> V3835 </td></tr><tr><td>超薄硝化棉coate芯片滑</td><td> gentel </td><td>路径</td></tr><tr><td>幻灯片印记（可选） </td><td>凝胶公司</td><td> WSP60-1 </td></tr><tr><td>沙克尔</td><td>费舍尔</td><td> 15-453-211 </td></tr><tr><td>离心分离</td><td> Eppendorf公司</td><td> 5804 000.013 </td></tr><tr><td>洗手盆/幻灯片幻灯片可拆卸支架染色菜</td><td>费舍尔</td><td> 08-812 </td></tr><tr><td>幻灯片孵化室/显微镜幻灯片中</td><td>费舍尔</td><td> 03-448-5 </td></tr><tr><td>布里杰35 W / V，30％水溶液</td><td> Acros Organics公司</td><td> AC32958-0025 </td></tr><tr><td>吐温20 </td><td>费舍尔</td><td> P337-100 </td></tr><tr><td>高碘酸钠（NAIO 4） </td><td>西格玛</td><td> 311448 </td></tr><tr><td> L-谷氨酸γ-肼</td><td>西格玛</td><td> G - 7257 </td></tr><tr><td>无水醋酸钠（CH 3 COONa） </td><td>西格玛</td><td> S2889 </td></tr><tr><td>牛血清白蛋白（BSA） </td><td> lampire生物实验室</td> &lt;TD&gt; 7500804 </td></tr><tr><td>磷酸盐缓冲液（PBS）（10X） </td><td>丹维尔科学</td><td> CP4390-48 </td></tr><tr><td> dylight 549共轭NeutrAvidin </td><td>热</td><td> 22837 </td></tr><tr><td>蛋白酶抑制剂鸡尾酒片</td><td>罗氏公司</td><td> 4693159001 </td></tr><tr><td> ChromPure人IgG，Fc片段</td><td>杰克逊Immunoresearch </td><td> 009-000-008 </td></tr><tr><td> ChromPure人IgG，整个分子</td><td>杰克逊Immunoresearch </td><td> 009-000-003 </td></tr><tr><td> ChromPure小鼠IgG，整个分子</td><td>杰克逊Immunoresearch </td><td> 015-000-003 </td></tr><tr><td> ChromPure小鼠IgG，Fc片段</td><td>杰克逊Immunoresearch </td><td> 015-000-008 </td></tr><tr><td> ChromPure兔IgG，整个分子</td><td>杰克逊Immunoresearch </td><td> 011-000-003 </td></tr><tr><td> ChromPure驴抗体，整个分子</td><td>杰克逊Immunoresearch </td><td> 017-000-003 </td></tr><tr><td>微阵列扫描仪</td><td> Tecan公司</td><td>的LS重装上阵</td></tr></tbody></table> 表2。在本协议所使用的设备和试剂的名单。

chemically-blocked antibody microarray for multiplexed high-throughput profiling of specific protein glycosylation in complex samples

在这项研究中，我们描述了一个用于在复用高通量抗体芯片与糖结合蛋白检测，允许特定蛋白质的糖基化分析的有效协议。蛋白质糖基化是最常见的翻译后修饰，对蛋白质的发现，导致多元化的修改的物理，化学，生物学特性和蛋白质。由于糖基化机制是特别容易受到疾病的进展和恶性转化，异常糖基化已被确认为癌症和其他疾病的早期检测标志物。然而，目前的方法来研究蛋白糖基化通常是在最普通的实验室或临床的设置和使用更实用的方法来研究蛋白糖基化需要过于复杂或昂贵。在这项研究中所描述的新的协议，使得与糖结合使用的化学阻断抗体芯片蛋白（GBP）检测，并显着减少的时间，成本和实验室设备，需要研究蛋白糖基化的要求。在此方法中，多个固定的糖蛋白特异性抗体直接打印到芯片的幻灯片和抗体的N-糖链被封锁。受阻，固定糖蛋白特异性抗体能够捕获和隔离从一个复杂的样品，直接应用到芯片幻灯片糖蛋白。聚糖检测，然后可以进行生物素标记的凝集素和其他Gbps的芯片幻灯片的应用，而具有约束力的水平可以决定使用Dylight 549-链霉。通过使用多个生物素标记的凝集素抗体面板和探测，这种方法可以有效的发展，在一个特定的人或动物样品中发现的不同的蛋白质糖基化的文件。 介绍蛋白糖基化，这是我S的 ​​最普遍的对蛋白质翻译后的修改，修改的物理，化学，生物特性的一种蛋白质，在1-6各种生物过程中起着根本性的作用。由于糖基化机制是特别容易受到疾病的进展和恶性转化已被公认为早期发现癌症和其他疾病的7-12生物标志物，异常糖基化。事实上，目前大多数肿瘤标志物，如三级分数的α-1，甲胎蛋白（法新社）13-15肝癌，胰腺癌16 CA199检测，有17个是所有异常的糖蛋白的糖基。然而，研究蛋白糖基化的方法已经复杂，不适合常规实验室和临床设置。 Chen 等最近发明了一种化学阻断抗体芯片1糖结合蛋白（GBP），高吞吐量的检测方法在一个复杂的样品18届复轮廓本土糖蛋白的糖基化。多个固定的糖蛋白特异性抗体的亲和力在此基础芯片的方法，捕捉和隔离从复杂的混合物，直接对芯片的幻灯片的糖蛋白，对每一个人捕获的蛋白聚糖GBPS测量。因为所有正常的抗体含有N-糖链最Gbps的，可以确认，这种方法的关键步骤是化学阻断抗体聚糖结合英镑。在此过程中，CI S-二醇组抗体聚糖被首先氧化成醛基使用醋酸钠缓冲液避光NAIO 4。醛基肼组的交联剂，4 - （4 N-MaleimidoPhenyl的）丁酸盐酸肼（MPBH），随后的二肽，半胱氨酸，甘氨酸，酰亚胺组，共轭共轭MPBH。从而被转换成笨重没有羟基，这阻碍了外源凝集素和其他Gbps的绑定捕获抗体，聚糖对抗体的顺式二醇组。这堵过程使Gbps和外源凝集素结合，只捕获蛋白聚糖。阻断后，这个化学，血清标本聚糖检测，通过使用不同的生物素标记的凝集素和Gbps的抗体芯片孵育，用Cy3-链霉亲和观察。抗体面板和探测多个外源凝集素的并行使用提供了在一个给定的样本18-20离散多种蛋白质糖基化型材。该方法已成功地应用于多个不同的实验室1，7，13，19-31。然而，影响稳定MPBH和半胱氨酸，甘氨酸，复杂性和扩展的过程，在这个方法的重复性，该方法的有效性和效率。在这个新的协议，我们更换一个更加稳​​定MPBH和半胱氨酸，甘氨酸试剂谷氨酸肼（GLU-肼），从而显着提高了简化的方法，重现性和缩短整个程序，以便它可以在一个工作日内完成。在这个新的协议，我们描述的详细程序的协议，可以很容易地通过正常的实验室常规蛋白糖基化研究和技术，这是要获得可重复性和可重复的结果。

Watch this Scientific Journal Video about Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples at JoVE.com

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

在这项研究中，我们描述了一个复用高通量抗体芯片与外源凝集素的检测方法，可以在特定的蛋白质糖基化分析改进协议。该协议提供了新的可靠的试剂和显着降低，相比以前的程序的时间，成本和实验室设备的要求。

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 ...

chemically-blocked-antibody-microarray-for-multiplexed-high

Research

JoVE Journal

Biology

15.3K 次观看.  Institute for Hepatitis and Virus Research. 在这项研究中，我们描述了一个复用高通量抗体芯片与外源凝集素的检测方法，可以在特定的蛋白质糖基化分析改进协议。该协议提供了新的可靠的试剂和显着降低，相比以前的程序的时间，成本和实验室设备的要求。

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

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

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 ...

科研

生物学

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

Many types of organic phosphorus (P) molecules exist in environmental samples1. Traditional P measurements do not detect these organic P compounds since they do not react with colorimetric reagents2,3. Enzymatic hydrolysis (EH) is an emerging method for accurately characterizing organic P forms in environmental samples4,5. This method is only trumped in accuracy by Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy (31P-NMR) -a method that is expensive and requires specialized technical training6. We have adapted an enzymatic hydrolysis method capable of measuring three classes of phosphorus (monoester P, diester P and inorganic P) to a microplate reader system7. This method provides researchers with a fast, accurate, affordable and user-friendly means to measure P species in soils, sediments, manures and, if concentrated, aquatic samples. This is the only high-throughput method for measuring the forms and enzyme-lability of organic P that can be performed in a standard laboratory. The resulting data provides insight to scientists studying system nutrient content and eutrophication potential.

This protocol describes a high-throughput method of enzymatic hydrolysis that utilizes a microplate reader to measure and classify soil phosphorus as P monoesters, P diesters and inorganic P. Up to 96 samples can be measured at one time in a standard laboratory.

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 ...

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction1-4. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms1.
The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCG&beta;), which carries two N-glycans and four O-glycans 5. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.
Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans6,7. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), &beta;1-4 Galactosidase, and &beta;-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans8 . Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (&alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &alpha;1-3,6 Galactosidase, and &beta;1-3 Galactosidase ), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans.
SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 &mu;g), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.

Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

使用具体远藤和exoglycosidases糖基化蛋白质的鉴定和表征

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

双分子荧光互补流式细胞仪分析：高通量的定量方法研究蛋白质 - 蛋白质相互作用

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

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 ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression 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 ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 &#946;-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to &#946;-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 &#946;-lactamase selected at a clinically relevant dosage of ampicillin are provided.

经过一蛋白饱和诱变图书馆的功能评价规范利用高通量测序

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...

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.

基于蛋白质的继承在酿酒酵母中的高通量筛选

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

tchip-seq: 细胞专用表观体分析

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...