This work was supported by a grant from the NIH. The assays shown in Figure 1 was performed by Dr. Joseph Fasolo. We thank Dr. Rui Chen for helpful comments.

1.探针制备<ol><li>文化与净化用来检查与其他蛋白质的相互作用如下V5融合激酶探头: <ol><li>用新鲜挑染酵母菌株Y258（的MATa pep4-3，his4-580，ura3-53，leu2-3,112）含V5融合蛋白（从GATEWAY载体pYES-DEST52表示）。使用分离的蛋白质作为探针上的微阵列。板上完全合成 - 尿嘧啶（SC-URA）/ 2％葡萄糖/琼脂酵母和生长在30℃下从冷冻培养（-80℃的甘油原液）3天。 </li><li>从单个菌落接种起子培养（5-20​​毫升）和上摇动平台（220-250转）或车轮在30℃在钪浦/ 2％葡萄糖生长过夜。 </li><li>第二天早晨，接种将400ml钪浦/ 2％棉子糖与足够起子培养培养物至最终OD 600为0.1。 </li><li>成长到接种OD 600为0.6接着半乳糖诱导V5激酶福锡永构建表达通过添加3×酵母提取物/蛋白胨（YEP）补充有6％的半乳糖的溶液中，通过添加足够的由3倍稀释的诱导培养基，以便半乳糖的那个最终浓度为2％。 </li><li>诱导细胞在30℃下进行6小时的摇动平台上。使用2升锥形瓶中，以保证适当的通风。 </li><li>使用JA-10（或可比）转子通过纺丝400毫升细胞悬浮液在1000×g离心5分钟，在4℃下收获细胞。 </li><li>用50ml冰冷的PBS缓冲液并转移至50ml锥形管一次洗细胞。在冰冷的PBS缓冲液再次洗涤沉淀（没有清洁剂或其它添加剂），用于裂解和转移至2ml单元帽管裂解。 </li><li>在4℃旋转细胞在20,000×g离心1分钟以粒料和吸管离开缓冲区。 </li><li>管放置在冰上进行裂解步骤。 </li><li>裂解细胞（250-350微升粒料）用01:在1.5 1mm的氧化锆珠粒1体积细胞沉淀，珠子，和磷酸盐缓冲盐水（PBS）的裂解缓冲液，涡旋使用搅动平台3次将混合物在2分钟的间隔在4℃。 </li><li>离心裂解物在20,000×g离心在台式微量离心机，在4℃下10分钟。 </li><li>吸管上清液进聚碳酸酯高速离心管中并通过ulracentrufugation以150,000×g离心澄清30分钟，在4℃。 </li><li>转移的澄清的裂解物，以含有预洗涤的Ni 2+亲和树脂（〜100微升）的管和孵育在章动器2小时，在4℃下以捕获组氨酸（His）的6X标记V5-融合蛋白。 </li><li>为10分钟，在4℃下用洗涤缓冲液洗树脂三次。沉淀用台式离心机5分钟的树脂在1000 xg离心在4℃下并吸出上清液。加入新鲜的洗涤缓冲液，每次洗涤和洗涤过程中返回管中的章动器的搅拌。 </li><li>洗净后，STEps的是完整的，应用该洗涤树脂至新的G-25柱洗脱。 </li><li>申请25微升洗脱缓冲液中逐步的方式开始用100mM咪唑到500mM咪唑的50mM增量为总共9馏分。 </li><li>利用十二烷基硫酸钠聚丙烯酰胺凝胶测定所收集的级分（SDS-PAGE）和考马斯染色来确定最高度富集的馏分（秒）测定中使用，并添加甘油至30％，并储存于-80℃直至在使用检测。 </li></ol></li></ol> 2.在探测阵列注:请参阅本节的步骤2.6开始试验前的准备抗体溶液。 <ol><li>为了检测的相互作用，稀释V5-荧光团缀合的抗体（ 即 AlexaFluor 647），以在探针缓冲器260纳克/毫升和振荡调匀。 注:准备该抗体溶液30分钟，在使用前和将管上的nutat或者或轮，以确保完全混合和抗体的均匀悬浮液。 </li><li>稀释V5-融合蛋白探针，5-500微克/ ml的浓度范围。 注:优化每个蛋白质 - 蛋白质相互作用测定法中，使用探针的缓冲区。优化涉及增加更多的探针探测缓冲区。通常为10微克/毫升作为起点，并相应地根据信号强度调整。 </li><li>从冰箱中（-20℃）除去蛋白质芯片和带至4℃的冰箱在使用之前。 </li><li>直接添加封闭缓冲液到含有蛋白质micorarrays载玻片支架和盖顶用石蜡膜，防止泄漏。通过在50rpm在舞台上，在4℃振荡方框在封闭缓冲液中进行1小时的阵列。 </li><li>封闭后，将阵列转移到湿润的腔室冷却至4℃，并加入90微升稀释的探针直接到阵列表面。叠加的ARR带有凸起挺杆滑AYS孵育静态（无晃动）在加湿室中在4℃下1.5小时。 </li><li>在探针缓冲器在三个50ml锥形管中的每个1分钟洗阵列3次。滑动添加到含有足够预冷探针缓冲器完全包住滑动锥形管。允许升降滑轻轻滑落的蛋白质微阵列（不要强迫它关闭，因为这可能会导致损坏阵列表面）。 </li><li>作为前完成清洗（步骤2.5），并覆盖有凸起的防滑起苗后立即申请直接抗体溶液阵列。在加湿室中孵育阵列30分钟，在4℃。 </li><li>在台式离心机5分钟在室温下以800×g离心执行相同的洗涤步骤之前（3次1在探针缓冲min），而旋在50ml锥形管中。风干在滑动保持器阵列在黑暗中以扫描该阵列在647毫微米之前30分钟。 </li></ol>

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The authors declare that they have no competing financial interests.

提出最初执行使用85独特酵母蛋白激酶-V5融合蛋白进行比较跨越导致新激酶相互作用网络体内 1的识别酵母蛋白激酶不同及相关家族结合活性的协议。作为一个新兴的蛋白质组学分析技术，高通量（HTP）使用蛋白质微阵列依靠肽库，和真核和原核模式生物蛋白质组8,17筛选的发展;后来，这个实验平台应用于高等真核生物如植物和人类5,7。目前还没有商业上可得到的酵?...

执行蛋白质生物化学和结合的体内活性的全局分析的需要已导致对分析蛋白质-蛋白质相互作用（质子泵抑制剂）和整个蛋白质组1,3-8的翻译后修饰的新方法的发展。蛋白质微阵列被制造为包含抗体10,11-功能蛋白微阵列使用全长功能蛋白4-6,8,9，或分析蛋白质微阵列。它们被加工成含有高密度排列到显微镜载玻片用各种表面化学的蛋白，以促进所需的多种用于进行广泛的生化分析12的实验条件。硝化纤维素和醛的表面化学进行化学附着，通过赖氨酸或亲和力连接方法如镍螯合载玻片用于附His标签的蛋白质，谷胱甘肽对除其他13亲和力附件。 </p&gt; 使用功能的蛋白质微阵列来检测蛋白质-蛋白质相互作用，需要获得一个高质量的功能性蛋白质库14。S.酵母是适合于通过高拷贝亲和力配对产生这样的库标记蛋白构建与高通量色谱纯化技术。酵母基因组的绝大部分已测序，几乎整个蛋白质组可以从高拷贝质粒纯化被表达和生化分析12。一旦蛋白被获得并排列在384孔格式，它们被打印到载玻片允许快速并行多参数生化分析和生物信息学的询问8,14-16。蛋白质微阵列已经用于酶测定和相互作用与蛋白质，脂质，小分子和核酸中的许多其它应用。蛋白质的蛋白质组的表面上的辅助阵列使得它们适合于不同类型的分析检测的，包括免疫亲和，表面等离子体共振，荧光技术及其他技术。此外，它允许精细控制的实验条件下在那里它可能是很难做到的体内 。 该协议的目的是要证明的适当使用的功能性蛋白微阵列来检测蛋白质 - 蛋白质相互作用。此应用程序的使用的兴趣高度纯化的分析物（蛋白质）的蛋白质结合活性的高通量平行生化分析。 C-末端（羧基末端）标记的感兴趣的V5融合诱饵蛋白从在酵母菌株的蛋白质纯化优化的高拷贝质粒制备。 C-末端标记确保了全长蛋白质被翻译。在这项研究中使用的蛋白质是Tda1-V5融合蛋白激酶，其使用镍亲和树脂经由His6X标签纯化。该Tda1-V5融合construct为通过串行洗脱使用咪唑梯​​度洗脱最高度富集的馏分在测定法中使用纯化。

<table><tbody><tr><td>Petri Dishes</td><td>VWR</td><td>NC-10747</td><td>or comparable</td></tr><tr><td>Bacto yeast extract</td><td>Difco</td><td>0217-17</td><td></td></tr><tr><td>Bacto peptone</td><td>Difco</td><td>0118-17</td><td></td></tr><tr><td>Bacto agar</td><td>Difco</td><td>0140-01</td><td></td></tr><tr><td>Dextrose</td><td>Sigma</td><td>D9434</td><td></td></tr><tr><td>Raffinose</td><td>Sigma</td><td>R0514</td><td></td></tr><tr><td>Galactose</td><td>Sigma</td><td>G0750</td><td></td></tr><tr><td>Sc-Ura drop out media</td><td>MP Bio</td><td>114410622</td><td></td></tr><tr><td>Small Culture tubes</td><td>VWR/Fisher</td><td></td><td></td></tr><tr><td>Large Erlenmeyer Flask</td><td>VWR/Fisher</td><td></td><td></td></tr><tr><td>Centrifuge JA-10 rotor</td><td></td><td></td><td></td></tr><tr><td>Centrifuge tubes (500 ml)</td><td></td><td></td><td></td></tr><tr><td>Falcon tube (50 ml)</td><td>VWR/Fisher</td><td>21008-951</td><td></td></tr><tr><td>PBS Tablets</td><td>Sigma</td><td>P4417</td><td></td></tr><tr><td>FastPrep Tubes (2 ml screw cap tube)</td><td></td><td></td><td></td></tr><tr><td>FastPrep Machine</td><td>MP Biomedicals</td><td>116004500</td><td></td></tr><tr><td>Zircon Beads</td><td>BioSpec</td><td>11079105</td><td></td></tr><tr><td>Triton X100</td><td>Sigma</td><td>P4417</td><td></td></tr><tr><td>DTT</td><td>Sigma</td><td>D9779</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M8266&amp;nbsp;</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S3014&amp;nbsp;</td><td></td></tr><tr><td>Imidazole (pH = 7.4)</td><td>Sigma</td><td>I5513&amp;nbsp;</td><td></td></tr><tr><td>PMSF</td><td>Sigma</td><td>93482</td><td></td></tr><tr><td>Complete Inhibitor Cocktail (EDTA Free)</td><td>Roche</td><td>11873580001</td><td></td></tr><tr><td>Phosphatase inhibitor cocktail 1</td><td>sigma</td><td>P2850</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A2153&amp;nbsp;</td><td></td></tr><tr><td>Tween-20</td><td>Sigma</td><td>P1379&amp;nbsp;</td><td></td></tr><tr><td>ATP</td><td>Sigma</td><td>A1852&amp;nbsp;</td><td></td></tr><tr><td>Shaking Incubator (30 &amp;deg;C)</td><td></td><td></td><td></td></tr><tr><td>Nickel Affinity Resin</td><td>Life Technologies</td><td>R901-01</td><td></td></tr><tr><td>G25 column</td><td>GE life sciences</td><td>27-5325-01&amp;nbsp;</td><td></td></tr><tr><td>Nutator</td><td>VWR/Fisher</td><td></td><td></td></tr><tr><td>SDS-PAGE Gel (NuPage)</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Coomassie Blue Stain</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Monoclonal V5 Antibody</td><td>Life Technologies</td><td>R960-25</td><td></td></tr><tr><td>Alexa647 Tagged monoclonal V5 Antibody</td><td>Life Technologies</td><td>451098</td><td></td></tr><tr><td>Protein Microarrays Kit</td><td>Life Technologies</td><td>PAH0525013</td><td></td></tr><tr><td>Genepix Scanner</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Lifter Slips</td><td>Thomas Scientific</td><td></td><td>http://www.thomassci.com/Supplies/Microscope-Cover-Glass/_/LIFTER-SLIPS/</td></tr><tr><td>Lysis Buffer: 0.1% Triton X-100, 0.5 mM DTT,&lt;br/&gt; 2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 500 mM NaCl, 50 mM imidazole (pH 7.4), Complete protease inhibitor cocktail &amp;ndash; EDTA-free, Phosphatase inhibitor Cocktail 1, 1 mM PMSF&amp;nbsp; (add just prior to use)</td><td></td><td></td><td>Add the reagents to 1x PBS (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride; pH 7.4) to obtain the desired volume of lysis solution</td></tr><tr><td>Blocking Buffer: 1% bovine serum albumin (BSA), 0.1% Tween-20</td><td></td><td></td><td>Add the reagents to 1x PBS to obtain the desired volume of blocking buffer</td></tr><tr><td>Probe Buffer: 2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 0.5 mM DTT, 0.05% Triton X-100, 50 mM NaCl, 500 &amp;mu;M ATP (for kinases), 1% BSA</td><td></td><td></td><td>Add the reagent to 1x PBS to obtain desired volume of probe buffer</td></tr><tr><td>Wash Buffer: 0.1% Triton X-100, 500 mM NaCl, 0.5 mM DTT, 50 mM imidazole (pH 7.4),2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td>Add the reagent to 1x PBS to obtain desired volume of probe buffer</td></tr><tr><td>Elution Buffer: 0.1% Triton X-100, 0.5 mM DTT, 2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 500 mM NaCl, 50 &amp;ndash; 500 mM imidazole (pH 7.4), Complete protease inhibitor cocktail &amp;ndash; EDTA-free, Phosphatase inhibitor Cocktail 1, 1 mM PMSF (add just prior to use)</td><td></td><td></td><td>Before you begin, it is important to note the concentration gradient of imidazole (50 &amp;ndash; 500 mM) and to create separate tubes for each concentration (it is advisable to create the interval using 50 mM increments). Add the reagent to 1x PBS to obtain desired volume of probe buffer</td></tr><tr><td>3x YEP +6% galactose: Dissolve 30 g of yeast extract and&amp;nbsp; 60 g of peptone in 700 ml of H2O, and autoclave. Add 300 ml of filter sterilized 20% galactose (wt/vol)</td><td></td><td></td><td>&amp;nbsp;Galactose should not be autoclaved</td></tr></tbody></table>

probing high-density functional protein microarrays to detect protein-protein interactions

含〜4200重组酵母蛋白的高密度功能蛋白微阵列检查使用含有用于读出一个V5表位标签的亲和纯化的酵母激酶融合蛋白激酶蛋白质 - 蛋白质相互作用。纯化激酶通过酵母菌株的高仿蛋白质生产带有含下GAL诱导启动子激酶-V5融合构建的质粒优化培养获得。酵母生长在限制性培养基用中性碳源为6小时，随后通过感应用2％半乳糖。接着，将培养物收获和激酶用标准亲和层析技术来获得用于在测定中使用高度纯化的蛋白激酶纯化。将纯化的激酶稀释激酶缓冲到一个适当的范围内用于测定和蛋白微阵列被阻断之前与蛋白质微阵列杂交。该杂交后，阵列探测用单克隆V5 antibODY鉴定蛋白质结合的由激酶-V5蛋白。最后，该阵列是利用标准的微阵列扫描仪扫描，并且数据被提取用于下游信息学分析1,2，以确定高置信度设置蛋白质相互作用的体内下游验证。

使用标准的芯片读取器，以评估Tda1-V5蛋白激酶融合构建体对含有约4200独特S.酵母功能性蛋白微阵列诱饵蛋白质的蛋白质-蛋白质相互作用的活性，观察酵母 GST-融合蛋白。与Genepix软件进一步审讯发现多种结合不同强度的活动。的亲和力从单克隆V5-荧光团缀合的抗体结合至其靶（V5-激酶）导出的信号的梯度强度衡量。 ProCat评分算法2用于鉴定蛋白质-蛋白质相互作用跨两个单独的...

Watch this Scientific Journal Video about Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions at JoVE.com

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

使用含有几乎整个S.蛋白质芯片酵母蛋白质组被探查以十万计的蛋白质-蛋白质相互作用中并行快速无偏询问。这种方法可以用于蛋白质 - 小分子，翻译后修饰，并在高通量其它测定。

探讨高密度功能蛋白微阵列来检测蛋白质 - 蛋白质相互作用

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

probing-high-density-functional-protein-microarrays-to-detect-protein

Research

JoVE Journal

Biology

8.0K 次观看.  Stanford University. 使用含有几乎整个S.蛋白质芯片酵母蛋白质组被探查以十万计的蛋白质-蛋白质相互作用中并行快速无偏询问。这种方法可以用于蛋白质 - 小分子，翻译后修饰，并在高通量其它测定。 

视频: Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

解决由蓝变性PAGE和蛋白质相关剖析亲和纯化的蛋白复合物

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

科研

生物化学

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

低亲和力受体-配体相互作用的细胞外蛋白微阵列技术

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

比较方法来表征景观的宿主 - 病原体蛋白 - 蛋白相互作用

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

免疫与感染

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

使用多路复用共免疫沉淀对蛋白质相互作用网络动力学进行量化

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

微图案的表面上的蛋白质-蛋白质相互作用的体内检测

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

生物学

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 ...

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

的蛋白质相互作用的合作伙伴在哺乳动物细胞中使用SILAC-免疫定量蛋白质组学鉴定

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

鉴定蛋白质相互作用的网站使用多肽阵列

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

全基因组蛋白质相互作用通过蛋白质片段互补法（PCA）的活细胞筛选

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

Protein-protein Interfaces

Many proteins form complexes to carry out their functions, making protein-protein interactions (PPIs) essential for an organism&#39;s survival. Most PPIs are stabilized by numerous weak noncovalent chemical forces. The physical shape of the interfaces determines the way two proteins interact. Many globular proteins have closely-matching shapes on their surfaces, which form a large number of weak bonds. Additionally, many PPIs occur between two helices or between a surface cleft and a polypeptide chain or string.
Various computational and biochemical methods are used to study protein interfaces. Laboratory methods, such as affinity purification, mass spectrometry, and protein microarrays, can be used to identify new interactions. Co-immunoprecipitation of proteins and yeast two-hybrid screening are widely used to provide evidence on whether two proteins interact in vitro.&#160; Computer programs can predict PPIs based on similar interactions found in other proteins by comparing protein sequences and three-dimensional structures. Other computational approaches, such as phylogenetic profiling, predict PPIs based on the coevolution of binding partners. Additionally, gene fusion analysis is used to predict interaction partners by finding protein pairs that are fused in the genome of other organisms.&#160;
Proteins typically interact with multiple partners either at the same or different times, and they may contain more than one interaction interface.&#160; Many proteins form large complexes that perform specific functions that can only be carried out by the complete complex. In some cases, these protein interactions are regulated; that is, a protein may interact with different partners based on cellular needs. Further computational and statistical analyses sort such interactions into networks, which are curated in online interactome databases. These searchable databases enable users to study specific protein interactions, as well as design drugs that can enhance or disrupt interactions at the interface.

Protein Complexes and Protein-Protein Interactions

蛋白质-蛋白质接口

Many proteins form complexes to carry out their functions, making protein-protein interactions (PPIs) essential for an organism&#39;s survival. Most PPIs ...