AF foi apoiado por uma bolsa a partir do Conselho Europeu de Investigação no âmbito do Sétimo Programa-Quadro da Comunidade Europeia (FP7 / 2007-2013) / acordo de subvenção CEI n ° 203413 e pelo Centro Minerva de Bio-híbrido systems.HA complexo e AI são suportadas pelo Dalia e Dan Maydan Fellowship para estudantes avançados de graduação na Universidade Hebraica de Jerusalém.

1. Criando uma matriz de Peptide <ol><li> Dividir a sequência da proteína alvo em parcialmente sobrepostos 10-20 resíduos péptidos. Variar a quantidade de sobreposição no experimento específico e os recursos do laboratório de realização, mas, em princípio, quanto mais tempo a sobreposição da melhor. Ao projetar os peptídeos ter em conta conhecidos elementos da estrutura secundária da proteína que pode ser responsável pela interação. </li><li> Encomende a matriz peptídeo projetado através de fornecedores comerciais. Aqui, o uso péptidos ligados covalentemente à matriz através do C-terminal. </li></ol> 2. Bloqueio de ligação não específica <ol><li> Adicione uma solução tampão 50 mM Tris ou fosfato contendo 0,05% de Tween 20 (TBST / PBST) e ajustar ao pH desejado com uma quantidade medida de HCl ou NaOH (para saber a força iónica preciso) e a força iónica desejada com NaCl . Aqui, usar um pH de 7,5 e uma força iónica de 150 mM. </li><li> Adicione uma solução de bloqueio de2,5% (w / v) de leite em pó desnatado em TBST / PBST. </li><li> Para impedir a ligação não específica, imergir a matriz em 5 ml de solução de bloqueio. Incubar a matriz durante 2-4 horas à temperatura ambiente ou durante a noite a 4 ° C num agitador. </li></ol> 3. A incubação com a Proteína <ol><li> Lava-se a matriz primeiro com 5 ml de solução de bloqueio durante 30 segundos e, em seguida, duas vezes com 5 mL de TBST / PBST, durante 5 min num agitador à temperatura ambiente. </li><li> Incubar a matriz lavada com 5 ml de solução de proteína marcada com His contendo 2,5% (w / v) de leite em pó desnatado para prevenir a ligação não específica. Aqui, utilizar 4,5 ^ M de solução de proteína (STIL 500-650) dissolvido na solução de bloqueio descrito. NOTA: Normalmente de 5-10 uM de proteína são usados ​​para o rastreio, mas a concentração de proteína pode ser mesmo tão baixa como 2-3 uM, dependendo da afinidade de ligação e as concentrações locais de péptidos que se ligaram na matriz (a eficiência da síntese). O tampão no qual a proteínaé dissolvido pode variar, mas é comum para diluir a proteína utilizando a mesma solução de bloqueio descrito acima. </li><li> Incubar a matriz na solução de proteína de 3-8 horas à temperatura ambiente ou durante a noite a 4 ºC. </li></ol> 4. A incubação com o anticorpo <ol><li> Lava-se a matriz três vezes com 5 mL de TBST / PBST. A primeira lavagem é de 30 seg seguido por duas lavagens de 5 min no agitador, à temperatura ambiente. </li><li> Incubar a matriz lavada com 5 ml de anticorpo conjugado com HRP diluída (1: 1500) em um tampão de incubação que contém os mesmos ingredientes nas mesmas concentrações como a solução de bloqueio, durante 1 h num agitador à temperatura ambiente. O anticorpo pode ligar-se quer a proteína alvo, ou a etiqueta. </li><li> Realizar experiências de controlo testar a interacção do anticorpo com os péptidos sobre a matriz, especialmente se a matriz contém péptidos da proteína alvo (por exemplo, para estudos de oligomerização) através da repetição do mesmo protocolo sem step 3, a incubação com a proteína. </li><li> Lava-se a matriz três vezes com 5 mL de TBST / PBST. A primeira lavagem é de 30 seg seguido por duas lavagens de 5 min no agitador, à temperatura ambiente. </li></ol> 5. Leitura da Matriz <ol><li> Realizar desenvolvimento de quimiluminescência com um kit de substrato western blotting ECL. </li><li> Realizar a detecção com um analisador de imagem luminescente. </li><li> Analisar os resultados. Certifique-se de que ambos os duplicados na matriz apresentam os mesmos sinais, por isso os resultados são confiáveis. Se a estrutura do parceiro de proteína é conhecido, pesquisa sobre a estrutura para os péptidos de ligação e procurar os locais de ligação. Mesmo os péptidos que estão longe na sequência podem estar próximos uns dos outros na estrutura terciária e criar um local de ligação. </li></ol>

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The authors have nothing to disclose.

Rastreio de matriz péptido é uma excelente ferramenta para identificar os locais de ligação de uma proteína alvo nos seus parceiros. Este ensaio é rápido e fácil de realizar e os resultados podem ser obtidos em um ou dois dias de trabalho. Rastreio de matriz péptido é versátil e pode ser usado para muitas finalidades. Ele pode ser usado para a caracterização de modificações pós-tradução, tais como a fosforilação e identificação de substratos de quinases. Por exemplo: a quinase FLT3, que está relac...

Interacções proteína-proteína mediar a maior parte dos processos na célula viva. Interacções proteína Deficiência pode resultar em doença, as interacções proteicas importantes alvos de drogas. Assim, é muito importante para compreender essas interações a nível molecular. Interacções entre proteínas são estudados usando uma variedade de técnicas que vão desde os ensaios celulares e bioquímicos para ensaios quantitativos biofísicas, e estas podem ser realizadas tanto com as proteínas de comprimento completo, com domínios de proteína ou com péptidos. Peptídeos servir como excelentes ferramentas para estudar interações protéicas. Isto é porque os péptidos podem ser facilmente sintetizadas e permitem que a focagem sobre um sítio de interacção específica de um lado e de vários alvos de proteína de uma forma de alto rendimento, por outro lado 1,2. Rastreio de matriz é um péptido rápido, fácil de executar método para a obtenção de uma grande quantidade de dados sobre as interacções de uma proteína alvo com numerosos parceiros num curto espaço de tempo 3. Ao contrário de outros métodos bioquímicos ou biofísicas para detectar e analisar interacções proteína-proteína, péptido rastreio matriz requer uma concentração muito baixa de proteína e pode detectar ligação muito fraca. Matrizes de péptidos pode ser utilizada para muitas aplicações em interacções péptido-proteína, tais como mapeamento de proteína-proteína ou sítios de interacção ligando-receptor 4, interfaces de homo- ou hetero-oligomerização, anticorpos que caracterizam epitopos 5, 6 estudar as actividades enzimáticas e de alto rendimento estrutura- relação atividade (SAR) estuda 7. Para uma análise aprofundada sobre a seleção matriz peptídeo ver Katz et al. 4 Vários tipos de matrizes de péptidos existem actualmente. Existem duas principais estratégias sintéticas para preparar matrizes de péptidos: síntese dos péptidos antes anexando-os ao suporte sólido, ou a síntese de péptidos directamente no suporte sólido, utilizando principalmente o SPOT 4,8 técnica. Oos péptidos são sintetizados sobre o suporte sólido geralmente por 9-fluorenilmetiloxicarbonilo (Fmoc) 8 química. Entre os esquemas sintéticos comuns são apego peptídeo através do terminal N (por exemplo, JPT Pepstar matrizes 9) e apego peptídeo através do terminal C (por exemplo, PEPSCAN matrizes pepchip 10, JPT pepspot matrizes 9 ​​e INTAVIS matrizes celluspot 11) 4. O suporte sólido pode variar e assim faz a química do acoplamento de péptidos a ele. Péptidos de Cis-terminados pode ser ligado a lâminas de vidro através do grupo tiol 12. O terminal N de um péptido pode ser ligado covalentemente a um grupo hidroxilo na membrana de celulose através de esterificação do aminoácido ligado 8. Aqui é apresentado um protocolo detalhado para o rastreio de matrizes de péptidos como um método para estudar as interacções proteína-proteína. A matriz que é utilizada a matriz Celluspots, que é uma micro-array contendo grande amount de pontos (duplicatas de até 384 pontos) em uma membrana de celulose pequena apoiados por uma lâmina de vidro. Isto permite trabalhar com volumes baixos de proteína e anticorpos e obtenção quantidade significativa de dados por única experiência. Essa matriz também contém alta densidade peptídeo que permite a detecção de baixa afinidade de ligação. A matriz foi utilizado para o mapeamento da interacção Stil-chfr, o que é altamente importante para o controlo da proliferação celular normal 13. Descontrolada interacção entre as duas proteínas pode levar ao desenvolvimento de cancro. Ao mapear essa interação encontramos o sítio de ligação específico e resíduos de 14 vinculativa. Isso abre o caminho para a concepção de inibidores racionais que inibem esta interação proteína-proteína.

<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

Interacções proteína-proteína mediar a maior parte dos processos na célula viva e o controlo da homeostase do organismo. Interacções proteína Deficiência pode resultar em doença, as interacções proteicas importantes alvos de drogas. Assim, é muito importante para compreender essas interações a nível molecular. Interacções entre proteínas são estudados usando uma variedade de técnicas que vão desde os ensaios celulares e bioquímicos para ensaios quantitativos biofísicas, e estas podem ser realizadas tanto com as proteínas de comprimento completo, com domínios de proteína ou com péptidos. Os péptidos servir como excelentes ferramentas para estudar as interacções proteína desde os péptidos podem ser facilmente sintetizadas e permitem que o foco em locais específicos de interacção. Peptídeo matrizes permitem a identificação dos sítios de interacção entre duas proteínas, bem como a despistagem de péptidos que se ligam a proteínas alvo para fins terapêuticos. Eles também permitem alta produtividade estudos de SAR. Para a identificação dos locais de ligação, um TYPIcal matriz peptídica normalmente contém parcialmente sobrepostos 10-20 resíduos de péptidos derivados a partir das sequências completas de uma ou mais proteínas associadas da proteína alvo desejado. O rastreio da matriz para a ligação da proteína alvo revela os péptidos de ligação, o que corresponde aos locais de ligação nas proteínas parceiras, em um método fácil e rápido usando apenas uma pequena quantidade de proteína. Neste artigo descrevemos um protocolo de triagem de matrizes de peptídeos para mapear os sítios de interação entre uma proteína-alvo e seus parceiros. A matriz péptido foi concebido com base nas sequências das proteínas parceiras, tendo em conta as suas estruturas secundárias. As matrizes utilizadas neste protocolo foram Celluspots matrizes preparadas por INTAVIS bioanalíticos Instruments. A matriz é bloqueada para evitar ligação inespecífica e depois incubadas com a proteína estudada. A detecção utilizando um anticorpo revela os péptidos de ligação correspondentes aos sítios específicos de interacção entre as proteínas.

STIL é uma proteína centrosomal altamente importante. Ele controla a divisão celular normal e proliferação celular 13,15 - 19. STIL interage com várias proteínas 18,20,21, e a maior parte das interacções ocorrer através da sua parte central, que é uma região intrinsecamente desordenados (IDR) 14. Concebemos uma matriz composta de péptidos derivados da proteína de ligação STIL chfr. Chfr é um supressor de tumor que é induzida em resposta ao estresse 22 mitóti...

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.

A identificação da proteína-proteína Locais de interacção Usando Peptide Arrays

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

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17.9K visualizações.  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.

Vídeo: 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.

Split-Ubiquitina Yeast Membrana Baseado Two-Hybrid (MITO) do sistema: uma ferramenta poderosa para identificação de interações proteína-proteína

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

Biologia

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)

Bimolecular de fluorescência de Complementação Assay (BiFC) para Interação Proteína-Proteína em células de cebola Utilizando o Gene Gun Helios

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.

Imagem interações proteína-proteína In vivo</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.

Mutagênese e Análise Funcional de canais iônicos Heterologously Expresso em células de mamíferos

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.

A fase líquida ensaio de captura de afinidade usando pérolas magnéticas para estudar interação proteína-proteína: O Exemplo Poliovírus Nanobody-

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.

Citometria de fluxo e de Complementação de fluorescência Bimolecular: A alta taxa de transferência método quantitativo para estudo de interação proteína-proteína

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.

Identificando interacção proteína-proteína em<em&gt; Drosophila</em&gt; Chefes Adulto por Tandem Affinity Purification (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.

Investigando interações proteína-proteína em células vivas usando a transferência de Bioluminescência Resonance Energy

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.

Especificidade Análise de Proteína Lisina Metiltransferases Usando 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 ...

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).

Genome-wide proteína-proteína Interaction Triagem por proteína-fragmento de Complementação Assay (PCA) em células vivas

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