Name: chemically-blocked antibody microarray for multiplexed high-throughput profiling of specific protein glycosylation in complex samples
Uploaded: 2012-05-04T13:00:00
Description: 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

这项工作是由肝炎病毒研究的研究所支持。

1。打印检测抗体芯片<ol><li>所有的抗体稀释至0.5毫克/毫升磷酸盐缓冲液，pH值7.2（PBS）的。 </li><li> 40μl每个抗体的分装成384井源板。 </li><li> ，Scienion sciFLEXARRAYER的微阵列加载到384井源板。 </li><li> 20 PATH芯片幻灯片加载到微阵列为目标。 </li><li>设置打印48个相同的子阵发现在一个9x9的模式（ 图1E，1F）一式三份，其中27个抗体和控制蛋白质微阵列。 </li><li>启动微阵列打印抗体芯片的?...

<ol>
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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 ...

Research

JoVE Journal

Biology

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

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

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

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 ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

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 ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

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

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

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

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

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

High-throughput Screening 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

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 ...