Agradecemos a los Dres. Steve Monte y Zhang Xiaoning para el 35S:: GFP pGlowbug la construcción y la Comunidad Hokensen Doctorado de Investigación en el laboratorio de CH Z. L&#39;s es apoyado por los EE.UU. National Science Foundation (IOB0616096 y MCB0744752). ZL es parcialmente financiado por la Universidad de Maryland Estación Experimental Agrícola.

I. plásmido preparación El ADN plásmido utilizado para el bombardeo debe ser de gran pureza y una concentración de 500ng/μl o mayor. Para BiFC que participan dos construcciones de plásmidos, 25 mg de cada plásmido se necesita. En otras palabras, una relación de 1:1 molar de los dos plásmidos se mezclan para dar lugar a 50 ug de plásmido ADN total de la preparación de los cartuchos. Tenga en cuenta que la adición de más de 50 mg de ADN puede causar la aglomeración de las partículas de oro y deben ser evitados. Kits comerciales de extracción de ADN plásmido se recomiendan para el aislamiento de ADN de plásmido. Vectores del plásmido debe ser relativamente pequeño en tamaño, tales como pUC19. Hemos clonado SEU y LUH cDNA en pUC19 y pUC19-Spyne SPYCE, respectivamente 3. Vectores binarios para la transformación mediada por Agrobacterium son demasiado grandes e inadecuado para el bombardeo. II. Cartucho de preparación Preparación del cartucho consiste en partículas de recubrimiento de oro con el ADN del plásmido y colocarlos en un tubo de plástico que posteriormente se corta en trozos media pulgada de largo, que se pueden cargar en los titulares de cartucho de pistola de genes. Preparación del cartucho se describe en gran detalle en un artículo separado Jove 4 y por lo tanto no se describe aquí. Para el bombardeo de las células de la cebolla, se recomienda mezclar 50 g de ADN plásmido con 12,5 mg de partículas de oro por la preparación, que produce alrededor de un 50 cartuchos a 1 g de oro DNA/0.25 mg / cartucho. Cada cartucho es de un solo disparo. Las partículas de oro pueden ser 0,6 m ó 1,0 m de diámetro (Cat # 1652262 # 1652263 o gato, BioRad). También recomendamos el uso de 0,5 mg / ml de solución de PVP para la preparación del cartucho. Para BiFC, combine los dos ADN plásmido de cada socio que interactúan putativo en la preparación de un mismo cartucho. Después de la preparación del cartucho, es importante para poner a prueba los cartuchos por el disparo de los cartuchos nuevos hechos a una presión de helio de 150 a 200 psi en un Parafilm cuadrados que se envuelve alrededor de una placa de Petri. Esto pondrá a prueba si las partículas de oro pueden ser impulsados ​​de manera eficiente de los cartuchos, usted debe ver las partículas de oro débil en el Parafilm. Esto le da una idea del área que cubrirá cada disparo. Usted puede notar diferentes cantidades de partículas de oro impulsó a la Parafilm a diferentes presiones de helio (en el rango de 150 a 200 psi). Si no hay una diferencia significativa, elegir el de menor presión para el bombardeo. III. La preparación de los tejidos de cebolla En el día de rodaje, despegue la piel exterior de una cebolla amarilla grande. Usando una hoja de afeitar limpias y afiladas, corte 4-5 capas profundas de un área rectangular de aproximadamente 2 x 8 cm. Sacar el tejido rectangular cebolla, deseche el exterior dos capas, corte el resto de las capas de la cebolla en trozos de 2x1.5 cm. Coloque los trozos de cebolla en una placa de Petri con papel filtro (papel Whatman 3MM cortado en círculos) humedecido con agua estéril. Cubrir la placa de Petri y que están dispuestos a ir. IV. Bombardeo <ol><li> La carga de los cartuchos en los soportes del cartucho redondo blanco. Cartuchos que contienen diferentes construcciones de plásmidos se deben cargar en los titulares de cartucho diferente. Cada titular de cartucho puede almacenar hasta 12 cartuchos de 12 tiros. </li><li> Inserte la batería y el revestimiento de barril fresca en la pistola de genes Helios. Asegúrese de que tanto el revestimiento de barril y el arma han respectivas juntas tóricas adjunto. </li><li> En primer lugar, insertar un soporte para el cartucho vacío en la pistola de genes, con la marca # 12 en el soporte del cartucho hacia la parte superior de la pistola. Titular de avance cartucho de una o dos veces para asegurarse de que está correctamente colocada. </li><li> Adjuntar pistola en el tanque de helio y ajustar la presión de 150 a 200 psi. </li><li> Use protección para los oídos y un arma de fuego un par de veces con el soporte del cartucho vacío para limpiar la cámara de la pistola y para asegurarse de que la presión es estable. </li><li> Retire el soporte del cartucho vacío e insertar un soporte para el cartucho cargado. Avanzar en el soporte del cartucho para que el cartucho que desea el fuego se coloca en el fondo (6:00) del soporte del cartucho. Disparar a un pedazo de cebolla aquí proporciona una forma de control negativo para la fluorescencia de fondo. </li><li> Coloque pedazos de la cebolla en el centro de un plato vacío de Petri con la capa más interna de la cebolla hacia arriba. Resto de línea de la pistola de genes de barril en una placa de Petri y disparar el arma pulsando el botón lateral, mientras que apretar el gatillo. La transferencia de la cebolla bombardeados con una placa de Petri que contiene el papel de filtro húmedo 3MM. </li><li> Avanzar a la siguiente cartucho y fuego un pedazo de cebolla diferentes. Repita este paso hasta cuatro a cinco piezas de la cebolla se disparó. Cada pieza de cebolla se disparó una sola vez. </li><li> Cambiar al titular de un cartucho cargado con diferentes cartuchos que contienen una combinación diferente plásmido plásmido o diferentes. Recuerde que para cambiar el revestimiento barrils bien para prevenir la contaminación. Dispara cuatro hasta cinco trozos de cebolla. </li><li> Después del bombardeo, devolver todas las cebollas a los platos de Perti. Cubrir las placas con las tapas. Envuelva con Parafilm para evitar la desecación. Incubar los trozos de cebolla a temperatura ambiente en la oscuridad durante 16-48 horas. </li><li> Apague la presión del gas y la liberación antes de desmontar la pistola de genes desde el tanque de helio. Desenroscar el barril de línea, y quitar el soporte del cartucho y los cartuchos usados ​​(o no). </li><li> Limpieza de cartucheras y fundas barril sumergiéndolas en un vaso con agua y jabón. Colocar el vaso en un baño de ultrasonido y sonicar durante 20 minutos. Después, enjuague con agua para eliminar todos los residuos de jabón. Ponga en remojo en el 70% de etanol durante una hora y luego séquelas con toallas de papel. </li></ol> V. Observación <ol><li> Después de la incubación bombardeado trozos de cebolla durante 16-20 horas a temperatura ambiente, se puede observar las buenas prácticas agrarias o de fluorescencia YFP utilizando un microscopio de fluorescencia, como el Observer.z1 Zeiss Axio utilizados en este estudio. </li><li> El uso de fórceps con los extremos planos que poco a poco la cáscara de una capa única célula de la epidermis interna de la cebolla (la capa que está directamente enfrente de la pistola durante el bombardeo). Colocar la cáscara en una gota de agua sobre un portaobjetos. Añadir cubreobjetos pero tenga cuidado para minimizar las burbujas. </li><li> En campo claro del microscopio, en primer lugar comprobar corriente citoplasmática para asegurarse de que las células están vivas. Para ello, exponen en la diapositiva a la luz brillante campo durante unos minutos para calentar el tejido de la cebolla y luego buscar plástidos en movimiento. Corriente citoplasmática no siempre es fácil de ver, así que no se desanime si no puede ver. </li><li> Para la detección de fluorescencia, excitación uso apropiado y filtros de detección de buenas prácticas agrarias o YFP. Es importante incluir controles negativos, tales como trozos de cebolla disparó con un plásmido que no fluorescente. Débiles señales amarillas pueden aparecer a partir de células heridos, las burbujas de aire, o los escombros. También es importante incluir controles positivos. Un plásmido que contiene el reportero expresa constitutivamente fluorescentes, como 35S:: GFP es un excelente control positivo. Señales visibles las buenas prácticas agrarias de las células epidérmicas de cebolla (Figura 1 A, D) indicó que los cartuchos, los tejidos de cebolla, y el despido pistola de genes están debidamente preparados y ejecutados. Posteriormente, se observaron interacciones entre SEU-pSPYNE y LUH pSPYCE en trozos de cebolla bombardeados con cartuchos que contienen una mezcla de 35S:: SEU-pSPYNE y 35S:: LUH-pSPYCE AND. Débil fluorescencia YFP se observó en el único núcleo (Figura 1 B, E), lo que indica la interacción física directa entre SEU y LUH que llevó a los fragmentos de YFP dividir juntos en el núcleo. Tenga en cuenta que la señal de YFP BiFC es significativamente más débil que la señal de las buenas prácticas agrarias del 35S:: GFP. </li></ol> Resultados representante En nuestro presente estudio, el 35S:: control de plásmido GFP emite fluorescencia positiva fuerte que llena toda la celda, incluyendo el núcleo y el citoplasma (Figura 1 A, D). La proteína GFP es lo suficientemente pequeño como para difundir en el núcleo, sin una señal de localización nuclear. Por el contrario, SEU y LUH son dos factores de transcripción Arabidopsis demostrado previamente para participar en una levadura de dos híbridos de ensayo 2. SEU-pSPYNE (SEU fusionado con el fragmento N-terminal de YFP) y LUH-pSPYCE (SEU fusionado con el fragmento N-terminal de YFP), a la vez demasiado grande para difundir en el núcleo, contienen señales de localización nuclear 2,5. La señal fluorescente YFP detectados en el núcleo de la célula de cebolla (Figura 1 B, E) indica que el SEU-pSPYNE y LUH pSPYCE-pueden interactuar en los núcleos de cebolla. No hay señal de fluorescencia se detecta en las cebollas bombardeados con la mezcla de plásmidos de pSPYNE vector plásmido que contenía el fragmento N-terminal de YFP y LUH-pSPYCE (Figura 1 C, F). La fluorescencia BiFC mediada YFP es significativamente más débil que la fluorescencia de GFP-mediado desde el 35S:: GFP plásmido. En nuestro estudio, también mostraron la localización subcelular diferente (comparar Figura 1 A con B). Además, el número de células fluorescentes es significativamente menor para BiFC, como BiFC sólo funciona cuando ambos plásmidos asociados con éxito entrar y se expresan en las células de la cebolla mismo. <img src="/files/ftp_upload/1963/1963fig1.jpg" alt="Figura 1" /> Figura 1. Fluorescente (A, B, C) ​​y de campo claro (D, E, F) las imágenes de las células de cebolla bombardeados con diferentes construcciones de plásmidos. (A) y (D), las células de cebolla bombardeados con 35S:: GFP. GFP se difunde en todo el citoplasma y núcleo. (B) y (E), las células de cebolla bombardeados con la misma mezcla molar del 35S:: SEU-pSPYNE (SEU fusionado con el fragmento N-terminal de YFP) y 35S:: LUH-pSPYCE (LUH fusionados al fragmento C-terminal ) del ADN del plásmido. Una interacción entre el SEU-pSPYNE y LUH pSPYCE-se muestra por un núcleo fluorescente. (C) y (F), un control negativo que muestra las células de cebolla bombardeados con mezclas de vector pUC19-Spyne y plasmi LUH-pSPYCEds. No se detecta la señal fluorescente.

<ol>
	<li>Hu, C. D., Chinenov, Y., Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+interactions+among+bZIP+and+Rel+family+proteins+in+living+cells+using+bimolecular+fluorescence+complementation.">Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation.</a> Mol Cell. 9 (4), 789-789 (2002).</li><li>Sitaraman, J., Bui, M., Liu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LEUNIG_HOMOLOG+and+LEUNIG+perform+partially+redundant+functions+during+Arabidopsis+embryo+and+floral+development.">LEUNIG_HOMOLOG and LEUNIG perform partially redundant functions during Arabidopsis embryo and floral development.</a> Plant Physiol. 147 (2), 672-672 (2008).</li><li>Walter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+protein+interactions+in+living+plant+cells+using+bimolecular+fluorescence+complementation.">Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation.</a> Plant J. 40 (3), 428-428 (2004).</li><li>Woods, G., Zito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+gene+gun+bullets+and+biolistic+transfection+of+neurons+in+slice+culture.">Preparation of gene gun bullets and biolistic transfection of neurons in slice culture.</a> J Vis Exp. , (2008).</li><li>Azhakanandam, S., Nole-Wilson, S., Bao, F., Franks, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SEUSS+and+AINTEGUMENTA+mediate+patterning+and+ovule+initiation+during+gynoecium+medial+domain+development.">SEUSS and AINTEGUMENTA mediate patterning and ovule initiation during gynoecium medial domain development.</a> Plant Physiol. 146 (3), 1165-1165 (2008).</li><li>Zhang, X. N., Mount, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+alternatively+spliced+isoforms+of+the+Arabidopsis+SR45+protein+have+distinct+roles+during+normal+plant+development.">Two alternatively spliced isoforms of the Arabidopsis SR45 protein have distinct roles during normal plant development.</a> Plant Physiol. 150 (3), 1450-1450 (2009).</li></ol>

<ol><li> Para los usuarios de primera vez, se recomienda la práctica de todo el procedimiento, desde la preparación del cartucho a la observación, con un único plásmido reportero constitutiva como 35S:: GFP o 35S:: GUS. Se utilizó un 35S:: GFP plásmido obtenido de los Dres. Steve Monte y Zhang Xiaoning 6 (Figura 1 A, D). Los cartuchos de inyección de genes de control en la venta RioRad (Cat. 165-2244) no son adecuados para las plantas. </li><li> Para BiFC, un control positivo es necesario, que debe constar de dos socios conoc...

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<td>Yellow onion</td>
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<td>Any supermarket</td>
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<td>Helios Gene Gun</td>
<td>&nbsp;</td>
<td>BioRad</td>
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<td>Fluorescent microscope with appropriate filters for GFP or YFP</td>
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bimolecular fluorescence complementation (bifc) assay for protein-protein interaction in onion cells using the helios gene gun

Investigación de la función de genes en diversos organismos se basa en el conocimiento de cómo los productos de los genes interactúan unos con otros en su ambiente celular normal. La complementación bimolecular de fluorescencia (BiFC) Ensayo<sup&gt; 1</sup&gt; Permite a los investigadores visualizar interacciones proteína-proteína en las células vivas y se ha convertido en una herramienta de investigación fundamental. Este ensayo se basa en la asociación facilitado de dos fragmentos de una proteína fluorescente (GFP), que son cada uno unido a un socio potencial de interacción proteína. La interacción de los dos socios de la proteína que facilitaría la asociación de la N-terminal y C-terminal del fragmento de la GFP, lo que lleva a la fluorescencia. Para los investigadores de plantas, células epidérmicas de cebolla son un sistema ideal de experimentación para la realización de la prueba BiFC debido a la facilidad en la obtención de tejidos y la preparación de la cebolla y la visualización directa de la fluorescencia con un mínimo de fluorescencia de fondo. La pistola de genes Helios (BioRad) se utiliza comúnmente para bombardear plásmido de ADN en células de cebolla. Se demuestra el uso de Helios pistola de genes para introducir construcciones de plásmidos durante dos interactuando<em&gt; Arabidopsis thaliana</em&gt; Factores de transcripción, Seuss (SEU) y homólogo Leunig (LUH)<sup&gt; 2</sup&gt; Y la visualización de sus interacciones mediadas por las células de cebolla BiFC en la epidermis.

Watch this Scientific Journal Video about Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun at JoVE.com

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

Este artículo muestra cómo utilizar correctamente el BioRad Helios pistola de genes para introducir ADN plásmido en las células epidérmicas de cebolla y la forma de prueba para las interacciones proteína-proteína en las células de la cebolla basa en el principio de fluorescencia de Complementación bimolecular (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 ...

bimolecular-fluorescence-complementation-bifc-assay-for-protein

Research

JoVE Journal

Biology

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

19.5K vistas.  University of Maryland. Este artículo muestra cómo utilizar correctamente el BioRad Helios pistola de genes para introducir ADN plásmido en las células epidérmicas de cebolla y la forma de prueba para las interacciones proteína-proteína en las células de la cebolla basa en el principio de fluorescencia de Complementación bimolecular (BiFC)

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

Gene-gun Transfection of Hippocampal Neurons

Gene-gun Transfección de las neuronas del hipocampo

Investigación

Biología

Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.  

We describe a method for preparing DNA coated gold bullets and demonstrate the use of such bullets to biolistically transfect neurons in cultured hippocampal slices.

Preparación de balas de cañón de genes y transfección biolística de neuronas en cultivo Cortar

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed ...

Bimolecular Fluorescence Complementation

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1)1,2. A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible3. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions4. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions5,6. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and F&ouml;rster resonance energy transfer (FRET)7. For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET experiments require the donor and acceptor to be of similar brightness and stoichiometry in the cell. In addition, one must account for bleed through of the donor into the acceptor channel and vice versa. Unlike FRET, BiFC has little background fluorescence, little post processing of image data, does not require high overexpression, and can detect weak or transient interactions. Bioluminescence resonance energy transfer (BRET) is a method similar to FRET except the donor is an enzyme (e.g. luciferase) that catalyzes a substrate to become bioluminescent thereby exciting an acceptor. BRET lacks the technical problems of bleed through and high background fluorescence but lacks the ability to provide spatial information due to the lack of substrate localization to specific compartments8. Overall, BiFC is an excellent method for visualizing subcellular localization of protein complexes to gain insight into compartmentalized signaling.

The subcellular localization of proteins is important in determining the spatio-temporal regulation of cell signaling. Here, we describe bimolecular fluorescence complementation (BiFC) as a straightforward method for monitoring the spatial interactions of proteins in the cell.

Fluorescencia de Complementación bimolecular

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as ...

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

Detección de interacciones de proteínas en la planta mediante un Gateway Compatible Complementación fluorescencia bimoleculares (BiFC) del sistema

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

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 ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Usar secuencias de detención SecM como una herramienta para aislar Ribosome polipéptidos enlazados

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

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 ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Interacciones proteína-proteína se visualizó por fluorescencia Bimolecular Complementación en protoplastos de tabaco y hojas

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

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 ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

Imágenes de fluorescencia con un nanómetros Precisión (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...