我们感谢EUROSARF向我们发送咨询酵母表达质粒。我们也感谢来自瑞安Labadens社论帮助。这项工作是由美国国立卫生研究院一/ NINDS补助金（R01NS070962）为CW支持

1。产生UAS-TAP标签的转基因果蝇<ol><li>产生pUAST-TAP标签的DNA结构。 <ol><li>决定（N-或C-末端）诱饵蛋白的哪一侧的TAP标记应被融合到，基于蛋白质的结构/功能。详细信息请参见讨论。 </li><li>亚克隆目的基因的cDNA编码区插入pUAST-NTAP或pUAST-CTAP载体的多克隆位点（MCS），以生成N-或C-末端标记的UAS-TAP转基因，分别。参见图1详细的地图和可用限制性酶切位点和读码框。 </li></ol></li><li>产生UAS-TAP标记的转基因果蝇。 <ol><li>产生转基因果蝇以下用P元素介导的插入11标准协议。市售许多注射服务。 </li><li>交叉神经GAL4驱动程序（ 即 BG380-GAL4）每个单独的转基因株系，并确定蛋白表达水平每行由免疫印迹（用过氧化物酶抗过氧化物酶抗体）和/或免疫（抗-TAP抗体）的。在一般情况下，用蛋白质表达水平是接近内源蛋白的转基因株系被推荐用于丝锥程序。详细信息请参见讨论。 </li><li> PerformGAL4/UAS-based拯救实验来证实在TAP标记的转基因的功能，如果可丧失功能的目的基因的突变体。选择转基因，可以基本上挽救突变体的表型为下面的TAP实验。 </li></ol></li></ol> 2。准备样本的TAP程序<ol><li>生成一个苍蝇的股票，同时携带一个神经元GAL4驱动程序（ 如 BG380-Gal4的），为了缓解扩张苍蝇样本的选择TAP标签的转基因。收集GAL4驱动程序并在极少数情况下，UAS-转基因交叉时，上面的组合会导致生存和成长不利的F1后代。&lt;/ LI&gt; <li>收集小规模样品和优化裂解条件，增溶TAP-标签蛋白。 <ol><li>使用非离子洗涤剂NP-40（0.1-1％），NaDOC（0.1-1％）和Triton X-100（0.05-0.5％）的组合的系列裂解缓冲液中。 见表1和讨论了解更多信息。 </li><li>在二氧化碳垫的顶部，使用＃5镊子从表达苍蝇在TAP基因解剖20成人头和收集他们在1.5ml试管测试每个裂解缓冲液条件。 </li><li>添加100 UL检测裂解液管，均质头摸摸上下用塑料杵，然后再添100 UL测试缓冲区。 </li><li>旋转磁头裂解物在21500×g离心10分钟（4℃），并分离离心分离后的上清液和沉淀。加入25微升2×SDS上样缓冲液，以分别将沉淀和10微升4×SDS上样缓冲液30微升上清液。 </li><li>煮沸2样品5分钟，并分析它们并排侧使用SDS-PAGE及随后的Western印迹与PAP抗体。由TAP-蛋白水平的上清液与沉淀的比值决定的溶解度。 </li></ol></li><li>在大规模制备样品<ol><li>扩大并收集成蝇。 <ol><li>展开neuronal-GAL4/UAS-TAP-transgene股票的瓶子和翻转，每3天，直到累计250瓶用于收集瓶。 </li><li>收集1-3日龄成蝇入50ml锥形管，把管子立即在液氮中的苍蝇深度冻结。存储苍蝇在-80°C冰箱。注意，苍蝇的体积必须不超过50毫升管的2/3。 </li></ol></li><li>收集苍蝇头（执行上粉状干冰顶部此步骤）。 <ol><li>取出预冷的筛子，并从-80°C冰箱的研钵和杵，并把它们放在干冰，最好里面一个大的冰桶。栈中的两处美国标准试验筛与上个25号Ë顶部和底部40号​​。 </li><li>采取冻结苍蝇出来，把它们在液氮中，并保持苍蝇在那里约10分钟。涡或摇晃试管大力从机构打破了头，腿和翅膀。 </li><li>把混合物顶端筛子，然后摇动筛大力按住两个筛子一起。筛分后，尸体会留在上面筛，飞头将被保留在底部筛板，和腿，翅膀，和其他碎片将坠落于干冰。分开两筛并仔细苍蝇头转移到冷砂浆。 </li></ol></li><li>均质苍蝇头<ol><li>干冰的顶部，研磨头与臼和杵粉粒，然后将粉末转移到预冷是在冰上有15毫升的玻璃Dounce型组织研磨。 </li><li>测量粉碎机的重量前后头部样​​品倒入，然后计算出头部多少SAMP乐晕死。总共有6-15克飞头将足以为每个TAP实验做出相应的调整，以蛋白质的表达水平。加入15毫升冰冷的匀浆缓冲液（在步骤2.2优化的裂解液）的粉末，然后与中风的间隙过大杵，直到它容易使杵上去和下来。保持玻璃研磨机上的冰在任何时候。 </li></ol></li><li>准备上清液进行咨询<ol><li>将匀浆转移至高速离心管中并旋转20分钟，〜50,000×g离心（4℃）。将上清转移到一个新的高速离心管中，重复离心一次。 </li><li>将上清转移至超速离心管并进行40分钟〜250,000×g离心旋转，以进一步清除上清液。上清液准备超速离心后的串联亲和纯化程序。 </li></ol></li></ol></li></ol> 3。 TAP纯化</p&gt; 以下各节均来自塞拉芬实验室技术咨询protocol 12 (http://web.as.uky.edu/Biology/faculty/rymond/BIO%20510/Bertran%20Seraphin%27s%20TAP%20page.pdf ） <ol><li>执行的IgG磁珠亲和纯化<ol><li>准备的IgG琼脂糖珠而样品被离心。在一个15毫升的Falcon管中，用10ml冷的IgG洗涤缓冲液洗涤400微升IgG抗体磁珠3次。对于每次洗涤，摇动管轻轻地，持续2分钟，然后停止旋转的有孔玻璃珠在1,000 xg下再持续2分钟。在第三次洗涤结束时，除去缓冲，并只留下珠粒在管。 </li><li>将澄清的上清液（约15毫升）小心地转移到15ml试管中含有IgG的珠子。孵化珠和脑裂解液混合，4℃一个nutator 2小时。 </li><li>建立一个干净的空微柱约15ml总量在寒冷的房间。通过稳步浇注混合成列装载的IgG珠的混合物，尽量不要任何陷阱气泡柱内。让珠定居在列和缓冲慢慢靠重力流排水。 </li><li>用10毫升冷的IgG洗涤缓冲液彻底清洗柱后，所有的脑细胞裂解液通过结算的IgG柱流过。重复洗涤2倍。注意:永远不要让空气中的珠干燥。 </li></ol></li><li>执行TEV酶切<ol><li>在第三次洗涤后，用10毫升TEV裂解缓冲液反复冲洗色谱柱。这一步准备的IgG珠的sequestrates诱饵复杂的TEV酶切。 </li><li>右侧前TEV缓冲区中的最后一滴欲滴出来，把帽子在柱的底部阻塞流，添加包含130个单位TEV酶的列1.3毫升TEV缓冲区中，然后安全地帽顶部列。确保该列在两端密封良好。 </li><li>旋转柱在18℃维持2小时，以允许TEV酶裂解的肽在TEV站点并释放蛋白复合WHI乐离开背后的蛋白质域肽绑定到的IgG琼脂糖珠。 </li></ol></li><li>执行钙调素磁珠亲和纯化<ol><li>制备的钙调蛋白珠，而IgG抗体磁珠孵育与TEV酶。在一个15毫升的Falcon试管3次，每次用10ml冷的钙调蛋白的结合缓冲液洗涤200μl的钙调蛋白珠。对于每一个洗，轻轻摇动试管一个nutator 2分钟，然后降速珠在1000×g离心2分钟。在第三次洗涤结束时，取出所有的缓冲液和仅保留在管中的小珠。 </li><li>在TEV孵化（步骤3.2.3）的结束，返回的IgG列回冷室，并设置它直线上升。让珠粒沉降10分钟。 </li><li>取下顶盖，然后将底盖，然后收集在15毫升猎鹰管1.3毫升TEV酶切产物。让缓冲区完全排出。添加一个额外的200微升TEV缓冲柱推出列的死体积，收集了F低中相同的管中。 </li><li>添加4.5毫升钙调素结合缓冲液和4.5微升1米氯化钙上面收集的1.5毫升TEV酶切产物。将氯化钙2用于滴定的EDTA在TEV缓冲液中。该6毫升混合物转移到含有钙调素珠的管和旋转管在4℃在章动器1小时。 </li><li>设立另一个干净的空微柱约10ml总量在寒冷的房间。加载钙调蛋白珠混合物的柱，并允许它通过重力排出。 </li><li>当所有的溶液已通过结算钙调素柱流出，洗柱2次，每次用10ml冷的钙调素的结合缓冲液中。注意:避免干扰钙调素珠，并尽量保持珠子尽可能平整的表面在洗。 </li></ol></li><li>从洗脱柱钙调素的饵料复杂。 洗涤后右，洗脱钙调素列有五个部分的200微升冷Calmodu林洗脱缓冲液。对每个级分，轻轻地加入200微升的洗脱缓冲液的柱，并收集与一个标记1.5ml离心管中的洗脱液。重复此4倍。 </li><li>分析通过SDS-PAGE的蛋白质复合<ol><li>取一小等分来自五个部分（约30微升），并加入SDS上样缓冲液。煮沸样品5分钟，并加载样品侧由端与一个梯度（4-15％）的SDS-PAGE凝胶上的蛋白质分子标记。 </li><li>后样品中的凝胶已经完全解决，停止电泳，染色凝胶与任何G-250为基础的敏感胶体考马斯亮蓝染色程序，如"青银"染色13。银染是可选的，但不是优选的，因为它不与随后的质谱分析完全兼容。在-80℃冷冻用于进一步的分析，如质谱法用于发现纯化的蛋白质复合物的分子身份存储洗脱液的其余部分。硒Ë讨论。 </li></ol></li></ol>

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串联亲和纯化（TAP）的方法提供了双纯化协议，允许通过两个独立的亲和纯化步骤的分离和蛋白质复合物的富集。在TAP标签的设计并不局限于所显示的内容在本协议中，其它蛋白结合结构域和图案也同样适用，如果缓冲液条件进行相应调整。其他的TAP标签的一个很好的例子是GS-TAP标签，G蛋白的结合和链霉亲和结合基序，由Giulio Superti-Furga小组力求在培养的哺乳动物细胞系18纯化蛋白复合物...

定义调解特定的生物过程中的分子途径或网络是生物医学研究的终极目标之一。飞遗传学家们严重依赖正向遗传学，尤其是修饰符遗传筛选（包括增强和抑制屏幕），以确定共同合作，与平行，或上游或目的基因的下游因素。然而，正向遗传学的屏幕，很多时候无法识别重要的基因，如果发生变异，导致杀伤力在早期发育阶段，或与基因功能冗余和补偿其失去功能只会造成细微瑕疵是很难得分。克服这一困难的一种方法是筛选直接的蛋白 - 蛋白相互作用。超过十年，生物化学方法，包括酵母双杂交，噬菌体展示，化学交联，联合知识产权，串联亲和纯化（TAP） 等越来越列表。已被用于研究蛋白质- 蛋白质相互作用。每种方法都有自己的一套优势和问候敏感性和特异性的弱点。其中，TAP方法可以检测物理相互作用下的近生理条件下，保留了特异性和一致性2，并包括延伸到高通量的能力，分析3,4。 最初是由Rigautand同事开发1在酵母中的TAP方法。在该方法中，目的蛋白质的表达用TAP标记。在TAP标签藏着两个独立的亲和力结合结构域:一个蛋白结合于IgG域和钙调素结合结构域。这两个结构域通过一个TEV（烟草蚀纹病毒）裂解位点隔开。这样的组合使得两个独立的两轮亲和纯化，以充分降低非特异性绑定和丰富具体的绑定1。对于这种情况下，TAP方法是一个非常强大的METHOd，来确定在给定的蛋白质的体内相互作用，虽然过表达外源蛋白可使其更易于与蛋白质通常不复杂的，其内源性的对应关联。由于它的发展时，TAP方法已经在许多其他系统，包括细胞培养为基础的系统5,6等体内模型系统应用6-9。在这里，我们描述了果蝇的咨询方法的适应性。我们首先生成pUAST-NTAP和pUAST-CTAP矢量以有利于克隆的TAP标记的融合到感兴趣的基因或N-或C-末端。在UAS-TAP标记的转基因然后在神经GAL4驱动器10的控制下，对神经系统表达。接着，将大量的成年苍蝇头将被收集，其具有神经细胞的高含量，并且容易从身体的其它部位根据大小不同冷冻后分离。成人头均化并清零bŸ连续离心，取上清液受下述TAP程序。

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			<td height="42" style="height:42px;width:307px;">U.S.A. standard test sieve No. 25</td>
			<td style="width:104px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">04-881-18&#160;</td>
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			<td height="42" style="height:42px;">U.S.A. standard test sieve No. 40</td>
			<td style="width:104px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">04-881-21</td>
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			<td height="21" style="height:21px;">IgG sepharose beads</td>
			<td>Pharmacia</td>
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			<td height="21" style="height:21px;">Econo-column 0.5 cm x 15 cm</td>
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			<td style="width:104px;">Sigma</td>
			<td style="width:119px;">P8340</td>
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identifying protein-protein interaction in drosophila adult heads by tandem affinity purification (tap)

利用果蝇 （果蝇）进行遗传筛选已在生物科学的进步提出了许多里程碑式的发现。然而，使用生化屏幕旨在扩大从遗传分析中获得的知识是最近才开发。在这里，我们描述的方法来净化蛋白复合物，随着兴趣的成年苍蝇头任何蛋白质的联系人。这种方法充分利用了果蝇 GAL4/UAS系统来表达融合在苍蝇体内一个神经元串联亲和纯化（TAP）的标签的诱饵蛋白，然后实现两轮净化使用类似于最初建立在一个咨询的过程酵母1，净化相互作用蛋白复合物。在此过程结束时，获得其分子的身份可以通过质谱法来确定多蛋白复合物的混合物。候选蛋白的验证将来自R受益esource和便于执行​​功能丧失的研究中的苍蝇。类似的方法可以应用到其它蝇组织。我们相信，基因操作和这个蛋白组学方法在果蝇模型系统的结合具有很大的潜力来解决在神经生物学领域及以后的基本问题。

在这里，我们证明了我们的努力物色HIGHWIRE相互作用的蛋白质在果蝇大脑。 HIGHWIRE（HIW）及其脊椎动物和无脊椎动物同系物是巨大的泛素连接酶，调节神经系统14的发育和修复。他们分享了一些高度保守的功能域。然而，它们的分子的行动是不完全清楚。在工作​​完成的蠕虫，蝇，鼠导致了当前的工作模式，HIW作为一个E3连接酶，并作为支架蛋白，以促进形成一个多亚基的泛素化复合物?...

Watch this Scientific Journal Video about Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP at JoVE.com

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

果蝇是著名的强大的遗传操作，但不能进行深入的生化分析其适用性。在这里，我们提出了一个TAP为基础的程序，从果蝇大脑识别任何利益蛋白质相互作用的合作伙伴。此过程有可能导致新的研究途径。

鉴定蛋白质 - 蛋白质相互作用的<em&gt;果蝇</em&gt;成人头由串联亲和纯化（TAP）

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

identifying-protein-protein-interaction-drosophila-adult-heads-tandem

Research

JoVE Journal

Biology

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

13.8K 次观看.  Louisiana State University Health Sciences Center.  果蝇是著名的强大的遗传操作，但不能进行深入的生化分析其适用性。在这里，我们提出了一个TAP为基础的程序，从果蝇大脑识别任何利益蛋白质相互作用的合作伙伴。此过程有可能导致新的研究途径。 

视频: Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

分离纯化果蝇</em磁性微球的排序>外周神经元

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

科研

生物学

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

由一个小型的双特异性亲和标签的推动正交蛋白质纯化

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

鉴定蛋白质相互作用的合作伙伴使用串联亲和纯化

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA纯化的染色质隔离（CHIRP）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

液相亲和捕获试验用磁珠研究蛋白质相互作用:脊髓灰质炎病毒的Nanobody范例

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

蛋白复合物的鉴定<em&gt;大肠杆菌</em使用顺序肽亲和纯化技术相结合串联质谱法

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

成绩单和代谢产物的分离纯化<em&gt;果蝇</em&gt;头

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

蛋白质纯化方法测定结合亲和力微尺度热泳

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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 ...

鉴定蛋白质 - 蛋白质相互作用的

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分离纯化果蝇外周神经元