Son compatibles AF fue apoyado por una subvención a partir del Consejo Europeo de Investigación en virtud del Séptimo Programa Marco de la Comunidad Europea (FP7 / 2007-2013) / acuerdo de subvención del CEI n ° 203 413 y por el Centro Minerva de Bio-híbrido systems.HA complejo y AI por la Dalia y Dan Maydan de Becas para estudiantes de grado avanzados en la Universidad Hebrea de Jerusalén.

1. Diseño de una matriz de péptidos <ol><li> Divida la secuencia de la proteína diana en parte, la superposición de 10-20 residuos de péptidos. Variar la cantidad de solapamiento en el experimento específico y los recursos del laboratorio que realiza, pero, en principio, cuanto mayor sea el solapamiento mejor. En el diseño de los péptidos tienen en cuenta los conocidos elementos de estructura secundaria de la proteína que pueden ser responsables de la interacción. </li><li> Ordene la matriz péptido diseñado a través de proveedores comerciales. Aquí, el uso de péptidos unidos covalentemente a la matriz a través de la C-terminal. </li></ol> 2. El bloqueo de la unión no específica <ol><li> Hacer una solución tampón de Tris 50 mM o fosfato que contiene 0,05% de Tween 20 (TBST / PBST) y ajustar al pH deseado con una cantidad medida de HCl o NaOH (con el fin de conocer la fuerza iónica precisa) y la fuerza iónica deseada con NaCl . Aquí, utilizar un pH de 7,5 y una fuerza iónica de 150 mM. </li><li> Haga una solución de bloqueo de2.5% (w / v) de leche desnatada en polvo en TBST / PBST. </li><li> Para evitar la unión no específica, sumerja la matriz en 5 ml de solución de bloqueo. Incubar la matriz durante 2-4 horas a temperatura ambiente o durante la noche a 4 ºC en un agitador. </li></ol> 3. La incubación con la proteína <ol><li> Lavar la matriz primero con 5 ml de solución de bloqueo durante 30 seg y luego dos veces con 5 ml de TBST / PBST durante 5 min en un agitador a temperatura ambiente. </li><li> Incubar la matriz se lavó con 5 ml de marcado con his solución de proteína que contiene 2,5% (w / v) leche desnatada en polvo para evitar la unión no específica. Aquí, utilizar 4,5 M de solución de proteína (STIL 500-650) disuelto en la solución de bloqueo descrito. NOTA: Generalmente 5-10 proteína M se utilizan para la proyección, pero la concentración de proteínas puede ser incluso tan bajo como 2-3 M, dependiendo de la afinidad de unión y las concentraciones locales de los péptidos que se unían en la matriz (la eficiencia de la síntesis). El tampón en el que la proteínase disuelve puede variar, pero es común para diluir la proteína usando la misma solución de bloqueo descrito anteriormente. </li><li> Incubar la matriz en la solución de proteína durante 3-8 horas a temperatura ambiente o durante la noche a 4 ºC. </li></ol> 4. La incubación con el anticuerpo <ol><li> Lavar la matriz tres veces con 5 ml de TBST / PBST. El primero de lavado es de 30 seg seguido por dos lavados de 5 min en un agitador a temperatura ambiente. </li><li> Incubar la matriz se lavó con 5 ml de anticuerpo conjugado con HRP diluido (1: 1500) en un tampón de incubación que contiene los mismos ingredientes en las mismas concentraciones que la solución de bloqueo, durante 1 hora en un agitador a temperatura ambiente. El anticuerpo se puede unir la proteína diana o en la etiqueta. </li><li> Realizar experimentos de control de comprobación de la interacción del anticuerpo con péptidos en la matriz, especialmente si la matriz contiene péptidos de la proteína diana (por ejemplo, para la oligomerización estudios) repitiendo el mismo protocolo sin step 3, la incubación con la proteína. </li><li> Lavar la matriz tres veces con 5 ml de TBST / PBST. El primero de lavado es de 30 seg seguido por dos lavados de 5 min en un agitador a temperatura ambiente. </li></ol> 5. Lectura de la matriz <ol><li> Llevar a cabo el desarrollo de quimioluminiscencia con un kit de sustrato ECL Western Blot. </li><li> Realizar la detección con un analizador de imágenes luminiscente. </li><li> Analizar los resultados. Asegúrese de que los dos duplicados de la matriz muestran las mismas señales, por lo que los resultados son fiables. Si se conoce la estructura de la proteína de pareja, buscar en la estructura de los péptidos de unión y buscar los sitios de unión. Incluso los péptidos que son mucho en la secuencia pueden ser muy juntos en la estructura terciaria y crear un sitio de unión. </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.

Detección matriz péptido es una excelente herramienta para la identificación de los puntos de unión de una proteína diana en sus socios. Este ensayo es rápido y fácil de realizar y los resultados se pueden obtener en uno o dos días hábiles. Screening matriz péptido es versátil y se puede utilizar para muchos propósitos. Se puede utilizar para la caracterización de modificaciones posterior a la traducción tales como la fosforilación y la identificación de sustratos de quinasas. Por ejemplo: la quinasa FLT...

Interacciones proteína-proteína median la mayoría de los procesos en la célula viva. Interacciones de proteínas con deficiencias pueden dar lugar a la enfermedad, por lo que las interacciones proteína objetivos importantes de drogas. Es por lo tanto muy importante entender estas interacciones a nivel molecular. Interacciones de proteínas se estudiaron usando una variedad de técnicas que van desde ensayos celulares y bioquímicos a ensayos biofísicos cuantitativos, y estos se pueden realizar ya sea con proteínas de longitud completa, con dominios de proteínas o con péptidos. Los péptidos sirven como excelentes herramientas para estudiar las interacciones proteína. Esto es porque los péptidos pueden ser sintetizados fácilmente y permiten que el centrarse en un sitio de interacción específica por un lado y en múltiples dianas proteicas en un alto rendimiento en el otro lado 1,2. Screening matriz péptido es un rápido, fácil de realizar método para obtener una gran cantidad de datos acerca de las interacciones de una proteína diana con numerosos asociados en un corto período de tiempo 3. A diferencia de otros métodos bioquímicos o biofísicos para detectar y analizar interacciones proteína-proteína, péptido matriz de cribado requiere una concentración muy baja de proteína y puede detectar una unión muy débil. Arrays de péptidos pueden utilizarse para muchas aplicaciones en las interacciones proteína-péptido como el mapeo de la proteína-proteína o sitios de interacción receptor-ligando 4, interfaces de homo o hetero-oligomerización, los anticuerpos que caracterizan epítopos 5, el estudio de las actividades enzimáticas 6 y de alto rendimiento estructura- relación actividad (SAR) estudia 7. Para una revisión en profundidad acerca de la detección matriz péptido ver Katz et al. 4 Actualmente, existen diversos tipos de matrices de péptidos. Hay dos principales estrategias sintéticas para la fabricación de matrices de péptidos: síntesis de los péptidos antes de adjuntarlos al soporte sólido, o síntesis de péptidos directamente sobre el soporte sólido, principalmente utilizando la técnica de 4,8 SPOT. Lalos péptidos se sintetizan en el soporte sólido por lo general por 9-fluorenilmetiloxicarbonilo (Fmoc) química 8. Entre los esquemas sintéticos comunes son el apego péptido a través del extremo N-terminal (por ejemplo, JPT matrices Pepstar 9) y el apego péptido a través de la terminal C (por ejemplo, PEPSCAN matrices pepchip 10, JPT matrices pepspot 9 y Intavis matrices celluspot 11) 4. El soporte sólido puede variar y también lo hace la química del acoplamiento de péptidos a la misma. Péptidos terminados en Cys pueden estar unidos a portaobjetos de vidrio a través del grupo tiol 12. El terminal N de un péptido puede estar unido covalentemente a un grupo hidroxilo en la membrana de celulosa a través de esterificación del ácido amino unido 8. Aquí presentamos un protocolo detallado para el cribado de arrays de péptidos como un método para el estudio de interacciones proteína-proteína. La matriz que utilizamos es la matriz Celluspots, que es un micro-array que contiene gran amount de manchas (duplicados de hasta 384 puntos) sobre una membrana de celulosa pequeña apoyados por un portaobjetos de vidrio. Esto permite trabajar con bajos volúmenes de proteína y anticuerpos y la obtención de cantidad significativa de datos por solo experimento. Esta matriz también contiene péptidos de alta densidad que permite la detección de baja afinidad de unión. La matriz se utilizó para el mapeo de la interacción STIL-CHFR, que es altamente importante para el control de la proliferación celular normal 13. Interacción incontrolada entre las dos proteínas puede conducir al desarrollo de cáncer. Mediante la cartografía de esta interacción se encontró el sitio de unión específico y residuos 14 de unión. Esto allana el camino para el diseño de inhibidores racionales que inhiben esta interacción proteína-proteína.

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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

Interacciones proteína-proteína median la mayoría de los procesos en la célula y el control vivir homeostasis del organismo. Interacciones de proteínas con deficiencias pueden dar lugar a la enfermedad, por lo que las interacciones proteína objetivos importantes de drogas. Es por lo tanto muy importante entender estas interacciones a nivel molecular. Interacciones de proteínas se estudiaron usando una variedad de técnicas que van desde ensayos celulares y bioquímicos a ensayos biofísicos cuantitativos, y estos se pueden realizar ya sea con proteínas de longitud completa, con dominios de proteínas o con péptidos. Los péptidos sirven como excelentes herramientas para estudiar las interacciones de proteína ya que los péptidos pueden ser sintetizados fácilmente y permitir que el centrarse en los sitios específicos de interacción. Arrays de péptidos permiten la identificación de los sitios de interacción entre dos proteínas, así como la detección de los péptidos que se unen a la proteína diana con fines terapéuticos. También permiten alto rendimiento estudios SAR. Para la identificación de sitios de unión, un tipificaCal matriz péptido por lo general contiene en parte superpuestos 10-20 residuos de péptidos derivados de las secuencias completas de una o más proteínas asociadas de la proteína diana deseada. La detección de la matriz para la unión de la proteína diana revela los péptidos de unión, que corresponden a los sitios de unión en las proteínas asociadas, en un método fácil y rápido usando sólo una pequeña cantidad de proteína. En este artículo se describe un protocolo para la detección de arrays de péptidos para el mapeo de los sitios de interacción entre una proteína diana y sus socios. La matriz de péptido está diseñado sobre la base de las secuencias de las proteínas asociadas, teniendo en cuenta sus estructuras secundarias. Las matrices utilizadas en este protocolo fueron Celluspots matrices preparadas por Intavis Bioanalíticos Instruments. La matriz está bloqueado para evitar la unión no específica y después se incubaron con la proteína estudiada. Detección usando un anticuerpo revela los péptidos de unión correspondientes a los sitios específicos de interacción entre las proteínas.

STIL es una proteína centrosomal altamente importante. Controla la división celular normal y la proliferación celular 13,15 - 19. STIL interactúa con varias proteínas 18,20,21, y la mayoría de las interacciones ocurren a través de su parte central, que es una región intrínsecamente desordenados (IDR) 14. Hemos diseñado una matriz compuesta de péptidos derivados de la proteína de unión STIL CHFR. CHFR es un supresor de tumor que se induce en respuesta a estrés mitótico <sup...

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.

La identificación de sitios de interacción proteína-proteína utilizando péptido 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 vistas.  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.

Video: 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 levadura membrana base de dos híbridos (MITO) del sistema: Una poderosa herramienta para identificar interacciones proteína-proteína

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

Investigación

Biología

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 complementación de fluorescencia (BiFC) Ensayo de interacción proteína-proteína en las células de cebolla con la pistola de genes 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.

Imágenes de interacciones 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.

Mutagénesis y análisis funcional de los canales iónicos heteróloga se expresa en células de mamífero

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.

Una afinidad líquido fase de captura de ensayo utilizando partículas magnéticas para estudiar la interacción proteína-proteína: el ejemplo de poliomyelits-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.

El análisis por citometría de Complementación fluorescencia Bimolecular: A High Throughput Método cuantitativo para estudiar la interacción 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.

La identificación de la interacción proteína-proteína en<em&gt; Drosophila</em&gt; Jefes adultas por Tandem Affinity purificación (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.

La investigación de las interacciones proteína-proteína en las células vivas utilizando la transferencia de energía de resonancia de bioluminiscencia

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.

Análisis de la especificidad de la proteína lisina metiltransferasas Usando SPOT Péptido 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).

Genoma toda la Proteína-proteína interacción Screening por Complementación de ensayo de proteínas-fragmento (PCA) en las células vivas

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