房颤是由欧洲研究理事会的欧盟第七框架计划下，开始资助项目（FP7 / 2007-2013）/ ERC资助协议N°203413和密涅瓦中心生物混合复杂systems.HA和AI支持由达莉亚和丹公司Maydan奖学金为高级学位的学生在耶路撒冷的希伯来大学。

1.设计一个多肽阵列<ol><li>划分目标蛋白的序列插入部分重叠的10-20残基的肽。变化重叠在特定实验的数量和执行实验室的资源，但原则上重叠的时间越长越好。当设计的肽考虑到在蛋白质帐户已知的二级结构元件，可以是负责的相互作用。 </li><li>以便通过商业供应商所设计肽阵列。这里，使用的肽共价结合到通过C末端的阵列。 </li></ol> 2.阻断非特异性吸附<ol><li>使含有0.05％Tween 20（TBST / PBST）中的50mM的Tris或磷酸盐缓冲溶液，并调节到所需的pH用HCl或NaOH的（为了知道精确的离子强度）的测量的量和所需要的离子强度用NaCl 。这里，可以使用pH为7.5和150mM的离子强度。 </li><li>使封闭液2.5％（重量/体积）的TBST / PBST中的脱脂奶粉。 </li><li>为了防止非特异性结合，将其浸入阵列在5ml封闭溶液。孵育所述阵列为2-4小时，在室温下或过夜，在4ºC振荡器上。 </li></ol> 3.与蛋白质孵化<ol><li>用5毫升封闭液30秒，然后两次用5毫升TBST / PBST中5分钟，在摇动器上在室温下的第一洗阵列。 </li><li>孵育洗涤阵列用5ml含2.5％（重量/体积）脱脂奶粉，以防止非特异性结合的His标记的蛋白质溶液。这里，使用4.5的蛋白溶液微米（STIL 500-650）溶解在所述的封闭溶液。 注意：一般5-10微米蛋白质被用于筛查，但蛋白质浓度可以甚至低至2-3微米，这取决于结合亲和力和局部浓度下结合的阵列上的肽（效率的合成）。缓冲器，其中蛋白质溶解可以改变，但通常使用如上所述的相同的封闭溶液稀释的蛋白质。 </li><li>孵育的蛋白溶液的阵列为3-8小时，在室温下或过夜，在4ºC。 </li></ol> 4.与抗体孵育<ol><li>用5毫升TBST / PBST中洗涤阵列三次。第一次洗涤是30秒，随后用两个5分钟的洗涤在摇床上于室温。 </li><li>孵育洗涤阵列用5ml稀释的HRP偶联的抗体（1：1500），在包含相同的成分在相同浓度的封闭溶液，1小时在摇床上于室温温育缓冲液中。该抗体可结合任一靶蛋白或标签。 </li><li>通过重复相同的协议，而不STE进行对照实验在阵列上测试该抗体的相互作用的肽，特别是如果该数组包含从靶蛋白（ 例如 ，用于低聚的研究）的肽第3页，与蛋白质孵育。 </li><li>用5毫升TBST / PBST中洗涤阵列三次。第一次洗涤是30秒，随后用两个5分钟的洗涤在摇床上于室温。 </li></ol> 5.读阵列<ol><li>开展化学发光法的发展与ECL免疫印迹底物试剂盒。 </li><li>用发光图像分析仪进行检测。 </li><li>分析结果。确保该阵列上的两个重复显示相同的信号，因此，结果是可靠的。如果蛋白伴侣的结构是已知的，搜索该结构的结合肽，并查找结合位点。甚至肽远的顺序可以并拢的三级结构，并创建一个结合位点。 </li></ol>

<ol>
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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+microcephaly+protein+STIL+interacts+with+CPAP+and+is+required+for+procentriole+formation.">The human microcephaly protein STIL interacts with CPAP and is required for procentriole formation.</a> Embo J. 30 (23), 4790-4804 (2011).</li><li>Vulprecht, J., David, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STIL+is+required+for+centriole+duplication+in+human+cells.">STIL is required for centriole duplication in human cells.</a> Journal of Cell Science. 125 (5), 1353-1362 (2012).</li><li>Campaner, S., Kaldis, P., Izraeli, S., Kirsch, I. 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H., Britan-Rosich, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+Vif&#8211;A3G+interaction+using+peptide+arrays:+A+basis+for+anti-HIV+lead+peptides.">Mapping the Vif&#8211;A3G interaction using peptide arrays: A basis for anti-HIV lead peptides.</a> Bioorganic &amp; Medicinal Chemistry. 21 (12), 3523-3532 (2013).</li><li>Katz, C., Benyamini, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+the+interaction+between+the+antiapoptotic+Bcl-2+family+proteins+and+the+proapoptotic+protein+ASPP2.">Molecular basis of the interaction between the antiapoptotic Bcl-2 family proteins and the proapoptotic protein ASPP2.</a> Proc Natl Acad Sci U S A. 105 (34), 12277-12282 (2008).</li><li>Katz, C., Zaltsman-Amir, Y., Mostizky, Y., Kollet, N., Gross, A., Friedler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Basis+of+the+Interaction+between+Proapoptotic+Truncated+BID+(tBID)+Protein+and+Mitochondrial+Carrier+Homologue+2+(MTCH2)+Protein:+KEY+PLAYERS+IN+MITOCHONDRIAL+DEATH+PATHWAY.">Molecular Basis of the Interaction between Proapoptotic Truncated BID (tBID) Protein and Mitochondrial Carrier Homologue 2 (MTCH2) Protein: KEY PLAYERS IN MITOCHONDRIAL DEATH PATHWAY.</a> Journal of Biological Chemistry. 287 (18), 15016-15023 (2012).</li><li>Rosenberg, M. 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The authors have nothing to disclose.

多肽阵列筛选是一个极好的工具，用于识别其合作伙伴靶蛋白的结合位点。该测定法是快速和容易地进行，其结果可以在一个或两个工作日内得到。肽阵列筛选是通用的，并且可以用于多种用途。它可以被用于表征交翻译修饰如磷酸化，并确定激酶的底物。例如：FLT3激酶，它与急性骨髓性白血病，磷酸化通过肽阵列筛选6发现含酪氨酸的肽底物。酪氨酸激酶抑制剂帕唑帕尼和拉帕替尼的抑...

蛋白 - 蛋白相互作用介导的大部分过程中的活细胞。受损的蛋白质相互作用可导致疾病，使得蛋白相互作用重要的药物靶标。因此，这是理解这些相互作​​用在分子水平上非常重要。蛋白质相互作用所使用的各种技术，从细胞和生物化学测定以定量的生物物理测定法的研究，并且这些可被执行或者与全长蛋白，用蛋白质结构域或肽。肽作为优秀的工具来研究蛋白质相互作用。这是因为肽可以容易地合成，并允许集中于特定的相互作用位点，一方面与多个蛋白质靶在另一方面1,2-以高通量方式进行。肽阵列筛选是一种快速，容易获得在短时间内3大量关于靶蛋白与许多伴侣的相互作用数据的执行方法。不像其他的生化或生物物理方法，用于检测和分析蛋白质 - 蛋白质相互作用，肽阵列筛选，需要非常低的蛋白质的浓度，并且可以检测到非常微弱的结合。肽阵列可用于多种应用中的肽-蛋白质相互作用，如蛋白-蛋白或受体-配体相互作用位点4，同源或异源寡聚化接口的映射，表征抗体的表位5，学习酶活6和高通量构筑物效关系（SAR）研究7。为深入审查有关肽阵列筛选看到卡茨等人 4 几种类型的肽阵列的当前存在的。有用于制备多肽阵列两大合成策略：合成肽它们附着于固相载体上的肽的，或合成直接在固体支持物上，主要是使用SPOT技术4,8-之前。该肽是由9-芴基（Fmoc）化学8在固体载体上合成通常。其中常见的合成方案是通过N端的肽连接（ 例如，JPT pepstar阵列9）和肽附着通过C末端（ 如 PEPSCAN pepchip阵列10，JPT pepspot阵列9和INTAVIS celluspot阵列11）4等。固体支持物可以有所不同，所以没有肽偶联到它的化学性质。半胱氨酸终止的肽可以通过硫醇基团12被连接到载玻片上。的N末端 ​​的肽可以通过氨基酸的酯化共价结合到纤维素上的膜具有羟基附8。 这里，我们提出了详细的方案用于筛选肽阵列作为用于研究蛋白质 - 蛋白质相互作用的方法。我们所使用的阵列是Celluspots阵列，它是一种含有大摩在微阵列UNT通过载玻片支撑在一个小的纤维素膜的斑点（高达384点的重复）的。这使蛋白质的低量和抗体的工作，并取得显著量为每一次实验的数据。该阵列还包含高肽密度可以检测低亲和力结合。该阵列被用于映射STIL-CHFR相互作用，这是用于控制正常细胞的增殖13非常重要。这两种蛋白之间的不受控制的相互作用可以导致癌症的发展。通过映射这种互动，我们发现了特异结合位点，并结合残基14。这种铺平了道路设计能抑制这种蛋白质 - 蛋白质相互作用理性抑制剂。

<table border="0" cellpadding="0" cellspacing="0" width="747">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Name of Reagent/ Equipment</td>
			<td style="width:231px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peptide array</td>
			<td>INTAVIS Bioanalytical Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">His-probe Antibody (H-3) HRP</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-8036 HRP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EZ-ECL Kit</td>
			<td>Biological industries</td>
			<td>20-500-120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Skim Milk</td>
			<td>Becton, Dickinson and Company</td>
			<td style="width:119px;">232100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LAS-3000 camera&#160;</td>
			<td>FUJI film</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

identifying protein-protein interaction sites using peptide arrays

蛋白 - 蛋白相互作用介导的大部分过程中的生物体的活细胞和控制的动态平衡。受损的蛋白质相互作用可导致疾病，使得蛋白相互作用重要的药物靶标。因此，这是理解这些相互作​​用在分子水平上非常重要。蛋白质相互作用所使用的各种技术，从细胞和生物化学测定以定量的生物物理测定法的研究，并且这些可被执行或者与全长蛋白，用蛋白质结构域或肽。肽作为优秀的工具来研究蛋白质相互作用，因为肽可以容易地合成，并允许针对特定的相互作用位点。多肽阵列使两种蛋白之间的相互作用位点的识别，以及筛选的靶蛋白结合用于治疗目的的肽。它们还允许高通量SAR的研究。对于识别的结合位点，一个typi的卡尔肽阵列通常包含部分地重叠，从所希望的靶蛋白的一种或多种伴侣蛋白的完整序列的10-20个残基的肽。筛选阵列对靶蛋白的结合揭示了结合肽，对应于在伴侣蛋白仅使用少量的蛋白质的结合位点，以容易和快速的方法。 在这篇文章中，我们描述了一个协议，用于筛选肽阵列映射的目标蛋白及其合作伙伴之间的互动网站。肽阵列是基于伴侣蛋白考虑到它们的二级结构的序列设计。在这个协议中使用的阵列制备了INTAVIS生物分析仪器Celluspots阵列。该阵列被阻断，以防止非特异性结合，然后与所研究的蛋白温育。使用抗体检测显示对应于该蛋白之间的特异性相互作用位点的结合的肽。

STIL是一个非常重要的中心体蛋白。它控制着细胞的正常分裂和细胞增殖13,15 - 19。 STIL有几种蛋白质18,20,21交互，而大部分的相互作用的发生，通过它的中心部分，这是一种本质上无序区（IDR）14。我们设计了一个由STIL结合蛋白CHFR衍生肽组成的数组。 CHFR]是诱导响应于有丝分裂应力22肿瘤抑制。因为CHFR的STIL结合结构域的结构是未知的时候，我们将所述蛋白质域到15...

Watch this Scientific Journal Video about Identifying Protein-protein Interaction Sites Using Peptide Arrays at JoVE.com

Identifying Protein-protein Interaction Sites Using Peptide Arrays

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 ...

identifying-protein-protein-interaction-sites-using-peptide-arrays

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JoVE Journal

Biology

17.9K 次观看.  The Hebrew University of Jerusalem. 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.

视频: Identifying Protein-protein Interaction Sites Using Peptide Arrays

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

基于分体式泛素膜酵母双杂交（神话）系统：一个强大的工具用于识别蛋白质 - 蛋白质相互作用

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

科研

生物学

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

双分子荧光互补（附设）检测蛋白质相互作用的洋葱细胞使用的Helios基因枪

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

成像蛋白质 - 蛋白质相互作用在体内</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

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 ...

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 ...

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

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

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

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

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. ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

研究蛋白质 - 蛋白质相互作用活细胞使用生物发光共振能量转移

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs.

Peptide arrays synthesized by the SPOT method can be used to analyze the substrate specificity of Protein lysine methyltransferases (PKMTs) and to define the substrate spectrum of PKMTs to understand their biological role. This protocol describes how to synthesize peptide arrays, methylate them with PKMTs, and analyze the results.

蛋白赖氨酸甲基转移使用SPOT肽阵列特异性分析

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays ...

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 ...