AF a été financé par une subvention à partir du Conseil européen de la recherche sous septième programme-cadre de la Communauté européenne (FP7 / 2007-2013) / accord de subvention du CER n ° 203413 et par le Centre Minerva pour Bio-hybride systems.HA complexe et AI sont pris en charge par Dalia et Dan Maydan bourse pour les étudiants de diplôme d&#39;études supérieures à l&#39;Université hébraïque de Jérusalem.

1. Concevoir un tableau Peptide <ol><li> Diviser la séquence de la protéine cible en se chevauchant en partie les résidus 10 à 20 peptides. Varier la quantité de chevauchement sur l&#39;expérience spécifique et les ressources du laboratoire performant, mais en principe, plus le chevauchement le mieux. Lors de la conception des peptides prennent en compte les éléments connus de la structure secondaire de la protéine qui peut être responsable de l&#39;interaction. </li><li> Commandez le réseau de peptides conçu par les fournisseurs commerciaux. Ici, l&#39;utilisation des peptides liés de façon covalente à la matrice par l&#39;intermédiaire du C-terminal. </li></ol> Non-spécifique 2. bloquer la liaison <ol><li> Ajouter une solution de tampon Tris 50 mM et de phosphate contenant 0,05% de Tween 20 (TBST / PBST) et ajuster au pH désiré avec une quantité mesurée de HCl ou de NaOH (pour connaître la force ionique précis) et de la force ionique désirée avec NaCl . Ici, en utilisant un pH de 7,5 et une force ionique de 150 mM. </li><li> Faire une solution de blocage de2,5% (p / v) de lait écrémé en poudre dans du TBST / PBST. </li><li> Pour éviter la liaison non spécifique, immerger la matrice dans 5 ml d&#39;une solution de blocage. Incuber la gamme de 2 à 4 h à la température ambiante ou une nuit à 4 ° C sur un agitateur. </li></ol> 3. L&#39;incubation avec la protéine <ol><li> Laver le premier réseau avec 5 ml de solution de blocage pendant 30 secondes et ensuite deux fois avec 5 ml de TBST / PBST pendant 5 min sur un agitateur à température ambiante. </li><li> Incuber la gamme lavé avec 5 ml de His-solution de protéines contenant 2,5% (p / v) de lait écrémé en poudre pour éviter la liaison non spécifique. Ici, en utilisant 4,5 M de solution de protéine (STIL 500-650) dissous dans la solution de blocage décrite. NOTE: Habituellement 5-10 protéine uM sont utilisés pour le dépistage, mais la concentration de protéine peut être même aussi bas que 2-3 uM, en fonction de l&#39;affinité de liaison et les concentrations locales des peptides qui se lient à la matrice (l&#39;efficacité de la synthèse). Le tampon dans lequel la protéineest dissous peut varier mais est commun pour diluer la protéine en utilisant la même solution de blocage décrite ci-dessus. </li><li> Incuber la matrice dans la solution de protéine de 3-8 heures à la température ambiante ou une nuit à 4 ° C. </li></ol> 4. Incubation avec l&#39;Anticorps <ol><li> Laver le tableau trois fois avec 5 ml de TBST / PBST. Le premier lavage est de 30 secondes, suivi de deux lavages de 5 min sur un agitateur à température ambiante. </li><li> Incuber la gamme lavé avec 5 ml d&#39;anticorps conjugué à HRP dilué (1: 1500) dans un tampon d&#39;incubation contenant les mêmes ingrédients à la même concentration que la solution de blocage, pendant 1 heure sur un agitateur à température ambiante. L&#39;anticorps peut se lier soit à la protéine cible ou l&#39;étiquette. </li><li> Réaliser des expériences de contrôle de tester l&#39;interaction de l&#39;anticorps avec des peptides sur le réseau, surtout si le tableau contient des peptides de la protéine cible (par exemple, pour oligomérisation études) en répétant le même protocole sans step 3, l&#39;incubation avec la protéine. </li><li> Laver le tableau trois fois avec 5 ml de TBST / PBST. Le premier lavage est de 30 secondes, suivi de deux lavages de 5 min sur un agitateur à température ambiante. </li></ol> 5. La lecture du tableau <ol><li> Effectuer le développement de chimiluminescence avec un kit de substrat Western blot ECL. </li><li> Effectuer la détection avec un analyseur d&#39;image luminescent. </li><li> Analyser les résultats. Assurez-vous que les deux exemplaires de la matrice montrent les mêmes signaux, de sorte que les résultats sont fiables. Si la structure de la partenaire de protéine est connue, la recherche sur la structure des peptides de liaison et de chercher les sites de liaison. Même les peptides qui sont bien dans la séquence peuvent être rapprochés dans la structure tertiaire et de créer un site de liaison. </li></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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The authors have nothing to disclose.

Dépistage du tableau peptide est un excellent outil pour identifier les sites de liaison d&#39;une protéine cible dans ses partenaires. Ce test est rapide et facile à réaliser et les résultats peuvent être obtenus dans un ou deux jours ouvrables. Dépistage du tableau peptide est polyvalent et peut être utilisé à de nombreuses fins. Il peut être utilisé pour caractériser les modifications de traduction soumettre comme la phosphorylation et l&#39;identification des substrats de kinases. Par exemple, la kinase...

Les interactions protéine-protéine médient la plupart des processus dans la cellule vivante. Les interactions entre protéines peut provoquer une baisse de la maladie, ce qui rend les interactions entre protéines cibles médicamenteuses importantes. Il est donc très important de comprendre ces interactions au niveau moléculaire. Les interactions protéiques sont étudiés en utilisant une variété de techniques allant de dosages biochimiques et cellulaires à des analyses quantitatives biophysiques, et ceux-ci peuvent être réalisés soit avec des protéines de pleine longueur, avec des domaines de protéines ou de peptides. Les peptides sont d&#39;excellents outils pour étudier les interactions entre protéines. En effet, les peptides peuvent être facilement synthétisés et permettent la mise au point d&#39;un site d&#39;interaction spécifique d&#39;une part, et sur ​​plusieurs cibles protéiques d&#39;une manière à haut débit, d&#39;autre part de 1,2. dépistage de réseau de peptides est un moyen rapide, facile à réaliser procédé pour obtenir une grande quantité de données sur les interactions d&#39;une protéine cible avec de nombreux partenaires dans un court laps de temps 3. Contrairement à d&#39;autres méthodes biochimiques ou biophysiques pour détecter et analyser les interactions protéine-protéine, le criblage de la matrice de peptide nécessite une très faible concentration de protéine et peut détecter de très faible liaison. matrices de peptides peuvent être utilisés pour de nombreuses applications dans les interactions peptide-protéine tels que la cartographie de la protéine-protéine ou des sites d&#39;interaction ligand-récepteur 4, les interfaces d&#39;homo- ou d&#39;hétéro-oligomérisation, des anticorps caractérisant épitopes 5, étudier les activités enzymatiques 6 et un débit élevé structure- relation de l&#39;activité (SAR) étudie 7. Pour un examen approfondi sur le dépistage du réseau de peptides voir Katz et al. 4 Plusieurs types de tableaux peptidiques existent actuellement. Il existe deux stratégies de synthèse pour la fabrication de grands ensembles de peptides: synthèse des peptides avant de les attacher au support solide, ou la synthèse de peptides directement sur ​​le support solide, en utilisant essentiellement la technique de 4,8 SPOT. Leles peptides sont synthétisés sur le support solide généralement par 9-fluorénylméthyloxycarbonyle (Fmoc) 8 chimie. Parmi les schémas de synthèse courantes sont l&#39;attachement peptide par l&#39;extrémité N-terminale (par exemple, les JPT PepStar tableaux 9) et la fixation de peptides grâce à l&#39;extrémité C-terminale (par exemple, des tableaux de PEPSCAN pepchip 10, JPT pepspot tableaux 9 et Intavis tableaux de celluspot 11) 4. Le support solide peut varier et il en va de la chimie du couplage peptidique pour elle. Peptides Cys-terminés peuvent être fixés à des lames de verre par l&#39;intermédiaire du groupe thiol 12. L&#39;extrémité N-terminale d&#39;un peptide peut être lié de manière covalente à un groupe hydroxyle sur la membrane de cellulose par estérification de l&#39;acide aminé fixé huit. Ici, nous présentons un protocole détaillé pour cribler des matrices de peptides en tant que méthode pour l&#39;étude des interactions protéine-protéine. Le tableau que nous avons utilisé est le tableau Celluspots, qui est un micro-array contenant grand amount de taches (doublons de 384 points) sur une petite membrane de cellulose pris en charge par une lame de verre. Cela permet de travailler avec de faibles volumes de protéines et d&#39;anticorps et d&#39;obtenir quantité importante de données par expérience unique. Ce tableau contient également la densité de peptide élevé qui permet la détection de faible affinité de liaison. La matrice a été utilisé pour cartographier l&#39;interaction STIL-CHFR, ce qui est très important pour le contrôle de la prolifération cellulaire 13 normal. Interaction non contrôlée entre les deux protéines peut conduire au développement d&#39;un cancer. En cartographiant cette interaction, nous avons trouvé le site de liaison spécifique et les résidus 14 de liaison. Cela ouvre la voie à la conception d&#39;inhibiteurs rationnels qui inhibent cette interaction protéine-protéine.

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	<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

Les interactions protéine-protéine médient la plupart des processus dans la cellule et le contrôle de l&#39;homéostasie de l&#39;organisme vivant. Les interactions entre protéines peut provoquer une baisse de la maladie, ce qui rend les interactions entre protéines cibles médicamenteuses importantes. Il est donc très important de comprendre ces interactions au niveau moléculaire. Les interactions protéiques sont étudiés en utilisant une variété de techniques allant de dosages biochimiques et cellulaires à des analyses quantitatives biophysiques, et ceux-ci peuvent être réalisés soit avec des protéines de pleine longueur, avec des domaines de protéines ou de peptides. Les peptides sont d&#39;excellents outils pour étudier les interactions entre protéines depuis peptides peuvent être synthétisés facilement et permettre la mise au point sur des sites d&#39;interaction spécifiques. Tableaux peptidiques permettent l&#39;identification des sites d&#39;interaction entre les deux protéines, ainsi que le dépistage de peptides qui se lient à la protéine cible à des fins thérapeutiques. Ils permettent également des études SAR haut débit. Pour l&#39;identification des sites de liaison, un Typical réseau de peptides contient habituellement se chevauchant partiellement 10-20 résidus des peptides dérivés des séquences complètes d&#39;un ou plusieurs protéines partenaires de la protéine cible souhaitée. La sélection de la gamme pour la liaison de la protéine cible révèle les peptides de liaison, correspondant aux sites de liaison dans les protéines partenaires, dans une méthode facile et rapide à l&#39;aide de seulement petite quantité de protéines. Dans cet article, on décrit un protocole de criblage de matrices de peptides pour la cartographie des sites d&#39;interaction entre une protéine cible et de ses partenaires. Le réseau peptidique est conçue sur la base des séquences des protéines partenaires compte tenu de leurs structures secondaires. Les tableaux utilisés dans ce protocole étaient Celluspots tableaux préparés par Intavis Instruments de bioanalyse. L&#39;ensemble est bloqué pour éviter une liaison non spécifique et ensuite incubé avec la protéine étudiée. La détection en utilisant un anticorps révèle des peptides de liaison correspondant à des sites d&#39;interactions spécifiques entre les protéines.

STIL est une protéine très importante centrosomale. Il contrôle la division cellulaire normale et la prolifération cellulaire 13,15 - 19. STIL interagit avec plusieurs protéines 18,20,21, et la plupart des interactions se produisent à travers sa partie centrale, qui est une région désordonnée intrinsèque (IDR) 14. Nous avons conçu un tableau composé de peptides dérivés de la protéine de liaison CHFR STIL. CHFR est un gène suppresseur de tumeur qui est induite en réponse ...

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.

Identifier les sites d&#39;interaction protéine-protéine à l&#39;aide de peptides tableaux

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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17.9K vues.  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.

Vidéo: 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-ubiquitine membrane à base de levure double hybride (MYTH) Système: Un outil puissant pour identifier les interactions entre protéines

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

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Biologie

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)

Bimoléculaire complémentation de fluorescence (BiFC) Dosage pour les interactions protéine-protéine dans les cellules d&#39;oignon Utilisation du pistolet à gènes 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.

Imagerie interactions protéine-protéine 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.

Mutagenèse et analyse fonctionnelle des canaux ioniques hétérologue exprimée dans les cellules de mammifères

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.

Un essai en phase liquide d&#39;affinité de capture utilisant des billes magnétiques pour étudier interaction protéine-protéine: Le poliovirus Exemple-Nanobodies

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.

L&#39;analyse par cytométrie de flux par fluorescence bimoléculaire complémentation: une méthode quantitative à haut débit pour étudier l&#39;interaction protéine-protéine

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.

Identifier les interactions protéine-protéine dans<em&gt; Drosophila</em&gt; Têtes de Tandem Affinity Purification adultes (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.

Étudier les interactions protéine-protéine dans les cellules vivantes en utilisant le transfert d&#39;énergie de résonance de bioluminescence

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.

Spécificité Analyse des protéines lysine Methyltransferases Utilisation 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).

L&#39;échelle du génome interaction protéine-protéine Le dépistage par Protein-fragment complémentation Assay (PCA) dans les cellules vivantes

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