El trabajo en nuestro laboratorio es apoyado por becas de los NIH, el USDA Instituto Nacional de Alimentación y la Agricultura, NSF, BARD, DOE, y FBS al VC

1. Expresión y la extracción de las proteínas <ol><li> Diferencialmente etiqueta de las proteínas de la prueba para su detección. Etiqueta de la proteína a ser inmovilizado en la membrana (ProIM) con una etiqueta de un tamaño más grande (por ejemplo, IVA), y la etiqueta no fusionados se puede utilizar como un control de inmovilizado negativo (ProIMnc). Etiqueta de la proteína a ser utilizado como una sonda soluble (PROSOL), ya sea con una grande o una pequeña etiqueta. Fusible de la misma etiqueta a un control adecuado de proteínas solubles en negativo (ProSOLnc) e incluir en el ensayo de interacción para confirmar la especificidad de la unión detectado. </li><li> Elegir el sistema de expresión de la proteína para expresar proteínas etiquetadas, es decir, E. coli, baculovirus, etc, dependiendo de la necesidad de modificaciones post traduccionales potencial y el rendimiento de las proteínas. </li><li> Extracto ProIM y nc ProIM del organismo de elección (paso 1.2) mediante SDS-PAGE de tampón de carga (20% de glicerol, 4% SDS, 20 mM Tris-HCl, pH6.8), que contiene un cóctel inhibidor de la proteasa. Salir de la suspensión de células a temperatura ambiente durante 15 minutos, añadir un 0,5% de 2-mercaptoetanol y se hierve durante 5 minutos. </li><li> Extracto de PROSOL y nc PROSOL del organismo de elección (paso 1.2) usando un estándar de protocolos 16. Estas proteínas deben ser fácilmente soluble en el tampón de unión (140 mM NaCl, 10 mM Tris-HCl pH 7,4, 2 mM EDTA, 2 mM DTT, 1% de BSA, 0,1% Tween 20) utilizado en el paso 4.1. </li><li> Determinar la concentración de las proteínas recombinantes utilizando un método estándar, como Bio-Rad kit de ensayo de la proteína. como Bio-Rad kit de ensayo de la proteína. Si el extracto crudo, en lugar de la proteína purificada, se utiliza para el ensayo, la estimación de la concentración de la proteína de interés en este extracto por densitometría de barrido de la banda correspondiente a un SDS-poliacrilamida gel teñido con azul brillante de Coomassie R-250 y el uso de conocidos concentraciones de BSA como referencia. </li></ol> 2. Inmovilización de ProIM y ProIMnc en la membrana <ol><li> Resolver los dos conjuntos de extractos, cada uno con 1 mg de ProIM y nc ProIM en un gel de poliacrilamida-SDS de acuerdo con el protocolo estándar 16. </li><li> Después de la electroforesis, el lugar de la transferencia que el gel en 100 ml de tampón de transferencia (60 mM Glicina, 10 mM Tris, el 0,0006% SDS, 20% MeOH) y se incuba con agitación suave durante 20 minutos a temperatura ambiente. </li><li> Electrotransferencia las proteínas contenidas en el gel a una membrana de nitrocelulosa de acuerdo con el protocolo estándar 16. </li></ol> 3. Re-plegamiento de las proteínas de membrana <ol><li> Después de electrotransferencia, se incuba la membrana durante 15 minutos en 15 ml de un buffer (30 mM Tris-HCl pH 7,4, 0,05% de Tween 20) con agitación suave para eliminar los restos de SDS. </li><li> Después de vaciar con cuidado, la transferencia de la membrana a 25 ml de tampón de desnaturalización (7 M clorhidrato de guanidina, 2 mM EDTA, 50 mM DTT, 50 mM Tris-HCl pH 8,3). y se incuba durante 2 horas a temperatura ambiente con agitación suave. (Nota: La membrana de nitrocelulosa se vuelve opaco cuando se incubaron en el tampón de desnaturalización). y se incuba durante 2 horas a temperatura ambiente con agitación suave. </li><li> La transferencia de la membrana a 25 ml de TBS (10 mM Tris-HCl, 150 mM NaCl, pH 7,4) e incubar con agitación suave durante 5 minutos (Nota: Durante este paso, la membrana recupera su color blanco original). </li><li> La transferencia de la membrana a 25 ml de tampón de unión (véase el paso 1.2) e incubar a 4 ° C con agitación suave durante la noche. </li></ol> 4. La prueba de las ProIM por PROSOL <ol><li> Corte la membrana en dos tiras, conteniendo ambos ProIM y nc ProIM. </li><li> Que las soluciones de hibridación PROSOL y ProSOLnc mediante la dilución de la preparación con 1.10 mg de cualquiera de PROSOL o ProSOLnc en 10 ml de tampón de unión libre. La transferencia de cada membrana en la solución de hibridación o PROSOL ProSOLnc e incubar durante 1,5 horas a temperatura ambiente con agitación suave. </li><li> Quitar las membranas de las soluciones de hibridación y enjuáguese la boca tres veces durante 15 minutos en cada una de TBS. </li></ol> 5. Visualización de interacción proteína-proteína mediante inmunotransferencia <ol><li> Bloquear la membrana con el 2,5% de leche desnatada en TBST (10 mM Tris-HCl, 140 mM NaCl, 0,05% de Tween 20, pH 7,4) durante 1 hora a temperatura ambiente. Diluir el anticuerpo primario (anti-strepII anticuerpo policlonal de conejo) en el 0,5% de leche desnatada en TBST a la concentración recomendada por el fabricante. </li><li> Coloque las membranas bloqueado en la solución de anticuerpos, y se incubó durante 1 hora a temperatura ambiente o por la noche a 4 ° C con agitación suave. </li><li> Enjuague las membranas en TBST 20 ml durante 15 minutos, y dos veces por 5 min a temperatura ambiente con agitación suave. </li><li> Diluir el anticuerpo secundario (anticuerpo anti-conejo IgG) conjugado con peroxidasa de rábano picante (HRP) en el 0,5% de leche desnatada en TBST a la concentración recomendada por el fabricante. Coloque las membranas en la secundaria de anticuerpossolución y se incuba durante 1 hora a temperatura ambiente con agitación suave. </li><li> Enjuague las membranas en TBST 20 ml durante 15 minutos, y dos veces por 5 min a temperatura ambiente con agitación suave. Después de que el enjuague final en TBS, visualizar la interacción proteína-proteína con un sustrato de quimioluminiscencia HRP (por ejemplo, Millipore Immobilon oeste de quimioluminiscencia sustrato HRP). </li><li> Sonda de las mismas membranas con el anticuerpo primario para la etiqueta fusionado a ProIM, seguido por el anticuerpo secundaria apropiada como se describe en los pasos 5.1 a 5.4. Visualizar ProIM y ProIMnc como se describe en el paso 5.5 para validar la identidad de las bandas obtenidas en el ensayo de proteínas de membrana superpuesta (paso 5.5). En la mayoría de los casos, no es necesario pelar, ya que la señal obtenida por este paso es mucho más fuerte que la señal residual obtenida en la etapa 5.5. </li></ol> 6. Los resultados representativos: La interacción ANK-MP fue observado por BiFC en las células epidérmicas del tabaco (Figura 1). Debido a que MP es una proteína muy insoluble cuando se expresa en bacterias o en las plantas, la proteína de la membrana superposición de ensayo se adoptó para validar esta interacción in vitro (fig. 1B). Extractos de proteínas que contiene 1 g de GST-MP (ProIM) o GST fusionadas (ProIM nc) se resolvieron por SDS-poliacrilamida electroforesis en gel, seguido por electrotransferencia a una membrana de nitrocelulosa. Cuando estos ProIMs se probaron con solubles ANK-strepII (PROSOL), GST-MP, pero no fusionados GST, exhibió unión (fig. 1B, carriles 1, 2, en comparación con los carriles 5, 6). Por otra parte, cuando el mismo conjunto de ProIMs se probaron con un ProSOLnc relación, es decir, la quinasa de Arabidopsis NADH citoplásmico etiquetados strepII (NADH3-strepII), no vinculante, no se observó, lo que demuestra la especificidad de la interacción ANK-MP (fig. 1B, carriles 3, 4). <img src="/files/ftp_upload/2961/2961fig1.jpg" alt="Figura 1"/> Figura 1. La unión específica de tabaco ANK a TMV MP in vivo e in vitro. (A) ANK-MP interacción de las células vivas del tabaco epidérmica detectada por BiFC. Fuerte señal de YFP fue reconstruido cuando MP y ANK, fusionado a C-terminal y N-terminal mitades de YFP, respectivamente, fueron coexpressed en la epidermis del tabaco siguientes microbombardment de sus genes de codificación. Esta señal BiFC acumulado en puntos lagrimales en la periferia de la célula, que son diagnósticos de plasmodesmos 15. (B) ANK-MP interacción in vitro detectada por superposición de ensayo de proteínas de membrana. Extractos de proteínas que contiene 1 g de GST-MP (ProIM) o no fusionados GST (ProIMnc) se resolvieron en un 15% SDS-gel de poliacrilamida, seguido por electrotransferencia a una membrana de nitrocelulosa. El GST-MPProIM GSTProIMnc y se incubaron con 0,5 mg / ml de ANK-strepII (PROSOL), y ANK vinculante fue detectado por el sondeo de la membrana con el anticuerpo de conejo anti-strepII policlonales, seguido de IgG anti-conejo + anticuerpo secundario conjugado con M HRP (calles 1 y 2). Ni GST-MPProIM GSTProIMnc ni interactuó con una proteína no relacionada, la cinasa citoplasmática Arabidopsis NADH, fusionado con la etiqueta strepII (ProSOLnc, carriles 3 y 4). La identidad de la banda observada en este ensayo fue confirmada por el sondeo de la membrana con el anticuerpo anti-GST (carriles 5 y 6). Cuando la membrana se trata con tampón de desnaturalización sin ser lavadas con buffer A, la unión de la GST-MP para ANK se pierde, mientras que las proteínas identificadas contenidas en el GST y GST-MP contining extractos de proteína reaccionaron con ANK-strepII, lo que demuestra la importancia de la etapa de 3,1 antes del proceso de desnaturalización (líneas 7 y 8).

<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, 789-798 (2002).</li><li>Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+localization+of+interacting+proteins+by+bimolecular+fluorescence+complementation+in+planta.">Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta.</a> J Mol Biol. 362, 1120-1131 (2006).</li><li>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=Design+and+implementation+of+bimolecular+fluorescence+complementation+(BiFC)+assays+for+the+visualization+of+protein+interactions+in+living+cells.">Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells.</a> Nat Protoc. 1, 1278-1286 (2006).</li><li>Shyu, Y. J. <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+Caenorhabditis+elegans+using+bimolecular+fluorescence+complementation+analysis.">Visualization of protein interactions in living Caenorhabditis elegans using bimolecular fluorescence complementation analysis.</a> Nat Protoc. 3, 588-596 (2008).</li><li>Citovsky, V., Gafni, Y., Tzfira, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localizing+protein-protein+interactions+by+bimolecular+fluorescence+complementation+in+planta.">Localizing protein-protein interactions by bimolecular fluorescence complementation in planta.</a> Methods. 45, 196-206 (2008).</li><li>Ohad, N., Shichrur, K., Yalovsky, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+analysis+of+protein-protein+interactions+in+plants+by+bimolecular+fluorescence+complementation.">The analysis of protein-protein interactions in plants by bimolecular fluorescence complementation.</a> Plant Physiol. 145, 1090-1099 (2007).</li><li>Zamyatnin, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+the+integral+membrane+protein+topology+in+living+cells.">Assessment of the integral membrane protein topology in living cells.</a> Plant J. 46, 145-154 (2006).</li><li>Citovsky, V., Knorr, D., Schuster, G., Zambryski, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+P30+movement+protein+of+tobacco+mosaic+virus+is+a+single-strand+nucleic+acid+binding+protein.">The P30 movement protein of tobacco mosaic virus is a single-strand nucleic acid binding protein.</a> Cell. 60, 637-647 (1990).</li><li>Heinlein, M., Epel, B. L., Padgett, H. S., Beachy, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+tobamovirus+movement+proteins+with+the+plant+cytoskeleton.">Interaction of tobamovirus movement proteins with the plant cytoskeleton.</a> Science. 270, 1983-1985 (1995).</li><li>Tomenius, K., Clapham, D., Meshi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+by+immunogold+cytochemistry+of+the+virus+coded+30+K+protein+in+plasmodesmata+of+leaves+infected+with+tobacco+mosaic+virus.">Localization by immunogold cytochemistry of the virus coded 30 K protein in plasmodesmata of leaves infected with tobacco mosaic virus.</a> Virology. 160, 363-371 (1987).</li><li>Ding, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+plasmodesmata+are+specific+sites+of+localization+of+the+tobacco+mosaic+virus+movement+protein+in+transgenic+tobacco+plants.">Secondary plasmodesmata are specific sites of localization of the tobacco mosaic virus movement protein in transgenic tobacco plants.</a> Plant Cell. 4, 915-928 (1992).</li><li>Meshi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+the+30+kd+protein+of+tobacco+mosaic+virus:+involvement+in+cell-to-cell+movement+and+dispensability+for+replication.">Function of the 30 kd protein of tobacco mosaic virus: involvement in cell-to-cell movement and dispensability for replication.</a> EMBO J. 6, 2557-2563 (1987).</li><li>Wolf, S., Deom, C. M., Beachy, R. N., Lucas, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Movement+protein+of+tobacco+mosaic+virus+modifies+plasmodesmatal+size+exclusion+limit.">Movement protein of tobacco mosaic virus modifies plasmodesmatal size exclusion limit.</a> Science. 246, 377-379 (1989).</li><li>Waigmann, E., Lucas, W. J., Citovsky, V., Zambryski, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+functional+assay+for+tobacco+mosaic+virus+cell-to-cell+movement+protein+and+identification+of+a+domain+involved+in+increasing+plasmodesmal+permeability.">Direct functional assay for tobacco mosaic virus cell-to-cell movement protein and identification of a domain involved in increasing plasmodesmal permeability.</a> Proc. Natl. Acad. Sci. USA. 91, 1433-1437 (1994).</li><li>Ueki, S., Spektor, R., Natale, D. M., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ANK,+a+host+cytoplasmic+receptor+for+the+Tobacco+mosaic+virus+cell-to-cell+movement+protein,+facilitates+intercellular+transport+through+plasmodesmata.">ANK, a host cytoplasmic receptor for the Tobacco mosaic virus cell-to-cell movement protein, facilitates intercellular transport through plasmodesmata.</a> PLoS Pathog. 6, e1001201-e1001201 (2010).</li><li>Coligan, J. E., Dunn, B. M., Ploegh, H. L., Speicher, D. W., Wingfield, P. T., Taylor, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , (2011).</li><li>Tzfira, T., Vaidya, M., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VIP1,+an+Arabidopsis+protein+that+interacts+with+Agrobacterium+VirE2,+is+involved+in+VirE2+nuclear+import+and+Agrobacterium+infectivity.">VIP1, an Arabidopsis protein that interacts with Agrobacterium VirE2, is involved in VirE2 nuclear import and Agrobacterium infectivity.</a> EMBO J. 20, 3596-3607 (2001).</li><li>Chen, M. H., Sheng, J., Hind, G., Handa, A., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+the+tobacco+mosaic+virus+movement+protein+and+host+cell+pectin+methylesterases+is+required+for+viral+cell-to-cell+movement.">Interaction between the tobacco mosaic virus movement protein and host cell pectin methylesterases is required for viral cell-to-cell movement.</a> EMBO J. 19, 913-920 (2000).</li></ol>

No hay conflictos de interés declarado.

Este enfoque es adecuado para las pruebas de interacciones proteína-proteína entre las combinaciones de las proteínas, cuando al menos uno de los cuales las proteínas se disuelve fácilmente en el tampón de unión, y se aplicó con éxito a otra combinación de proteínas 17,18. El iInteractions entre las proteínas que son insolubles en estas condiciones no pueden ser probados por el presente Protocolo. Además, el replegamiento de ProIM éxito es crítico para el ensayo. En...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Proteína kit de ensayo </td><td> Bio-Rad </td><td> 500-0001 </td></tr><tr><td> Proteinasa cóctel de inhibidores de </td><td> SIGMA </td><td> S8820 </td></tr><tr><td> Mini-Protean sistema </td><td> Bio-Rad </td><td> 165-8000 </td></tr><tr><td> Semi-seco Western Blot SD electrotransferencia sistema </td><td> Bio-Rad </td><td> 170-3940 </td></tr><tr><td> IgG anti-conejo conjugado con peroxidasa de rábano de anticuerpos </td><td> GenScript </td><td> A00098 </td></tr><tr><td> Anti-GST anticuerpo policlonal de conejo </td><td> GenScript </td><td> A00097 </td></tr><tr><td> Anti-strepII </td><td> GenScript </td><td> A00626 </td></tr><tr><td> Biotrace, nitrocelulosa NT membrana de transferencia </td><td> Pall Científico </td><td> 27377-000 </td></tr><tr><td> Immobilon occidental HRP sustrato quimioluminiscente </td><td> Millipore </td><td> WBKL S0 050 </td></tr></tbody></table>

protein membrane overlay assay: a protocol to test interaction between soluble and insoluble proteins in vitro

Las interacciones entre las proteínas de la validación de diferentes es de vital importancia para la investigación de sus funciones biológicas a nivel molecular. Hay varios métodos, tanto in vitro como in vivo, para evaluar la unión a proteínas, y por lo menos dos métodos que complementen las carencias de cada uno debe realizar para obtener conocimientos fiables. Para un ensayo in vivo, la complementación bimolecular de fluorescencia (BiFC) ensayo representa el enfoque más popular y menos invasivo que permite detectar interacción proteína-proteína en las células vivas, así como identificar la localización intracelular de las proteínas que interactúan 1,2. En este ensayo, no fluorescente mitades N-y C-terminal de la GFP y sus variantes están fusionados a las proteínas de prueba, y cuando las dos proteínas de fusión se unen debido a las interacciones de las proteínas a prueba, la señal fluorescente se reconstituye 3-6 . Debido a que su señal es fácilmente detectable por microscopía confocal de epifluorescencia o, BiFC se ha convertido en una poderosa herramienta de elección entre los biólogos celulares para el estudio de las interacciones proteína-proteína en tres células vivas. Este ensayo, sin embargo, a veces puede producir resultados falsos positivos. Por ejemplo, la señal fluorescente se puede reconstituir dos fragmentos de las buenas prácticas agrarias dispuestas hasta 7 nm de cada uno de otro por cerca de embalaje en un compartimiento subcelular pequeña, más bien que debido a las interacciones específicas 7. Debido a estas limitaciones, los resultados obtenidos de las tecnologías de células en vivo de imágenes deben ser confirmados por un enfoque independiente sobre la base de un principio diferente para la detección de interacciones entre proteínas. Co-inmunoprecipitación (Co-IP) o el glutatión transferasa (GST) pull-down ensayos representan tales métodos alternativos que se utilizan comúnmente para analizar interacciones proteína-proteína in vitro. Sin embargo, eEn estos ensayos, sin embargo, las proteínas de las pruebas debe ser fácilmente soluble en el búfer que supportsused para la reacción de unión. Por lo tanto, las interacciones específicas que implican una proteína insoluble no puede ser evaluado por estas técnicas. Aquí, se expone el protocolo para el análisis de la proteína de membrana superpuesta vinculante, lo que evita este problema. En esta técnica, la interacción entre las proteínas solubles e insolubles puede ser fiable a prueba, porque una de las proteínas es inmovilizado en una matriz de membrana. Este método, en combinación con los experimentos in vivo, tales como BiFC, ofrece un método fiable para investigar y caracterizar las interacciones entre las proteínas fielmente soluble e insoluble. En este artículo, la unión entre el virus del mosaico del tabaco (TMV) proteína de movimiento (MP), que ejerce múltiples funciones viral en la célula a célula de transporte 8.14, y una planta de interactor identificado recientemente celular, el tabaco ankyrin repetir que contienen proteínas (ANK ) 15, se demuestra mediante esta técnica.

Watch this Scientific Journal Video about Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro at JoVE.com

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Pruebas de interacción proteína-proteína es indispensable para la disección de la funcionalidad de las proteínas. Aquí, presentamos un<em&gt; In vitro</em&gt; Proteína-proteína ensayo de unión a la sonda una membrana de proteína inmovilizada con una proteína soluble. Este ensayo proporciona un método fiable para comprobar la interacción entre una proteína insoluble y de una proteína en una solución.

De proteínas de membrana de superposición de ensayo: Un protocolo de prueba de interacción entre las proteínas soluble e insoluble In vitro</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

protein-membrane-overlay-assay-protocol-to-test-interaction-between

Research

JoVE Journal

Biology

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

22.0K vistas.  State University of New York. Pruebas de interacción proteína-proteína es indispensable para la disección de la funcionalidad de las proteínas. Aquí, presentamos un In vitro Proteína-proteína ensayo de unión a la sonda una membrana de proteína inmovilizada con una proteína soluble. Este ensayo proporciona un método fiable para comprobar la interacción entre una proteína insoluble y de una proteína en una solución.

Video: Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Tinción de las proteínas en geles con Coomassie G-250, sin ácido disolvente orgánico y el acético

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

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 ...

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 ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

El uso de un robot para la cristalización de alto rendimiento de proteínas de membrana en mesofases lipídicas

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

In vitro metilación de ensayo para estudiar la proteína arginina metilación

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

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 ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Fluorescente verde a base de proteína Expresión Proyección de proteínas de membrana en<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

Complementación de luciferase imagen ensayo en hojas de Nicotiana benthamiana para determinar transitoriamente proteínas interacción dinámica

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...

Determination of Tripartite Interaction between Two Monomers of a MADS-box Transcription Factor and a Calcium Sensor Protein by BiFC-FRET-FLIM Assay

Protein-protein interactions are an integral part of all biological processes in the cells as they play a crucial role in regulating, maintaining, and amending cellular functions. These interactions are involved in a wide range of phenomena such as signal transduction, pathogen response, cell-cell interactions, metabolic and developmental processes. In the case of transcription factors, these interactions may lead to oligomerization of subunits, sequestering in specific subcellular contexts such as the nucleus, cytoplasm, etc., which, in turn, might have a more profound effect on the expression of the downstream genes. Here, we demonstrate a methodology to visualize in vivo tripartite interaction using Bimolecular Fluorescence Complementation (BiFC) based F&#246;rster Resonance Energy Transfer (FRET) involving Fluorescence Lifetime Imaging (FLIM). Two of the proteins selected for this demonstration interact as BiFC partners, and their reconstituted fluorescence activity is used to assay FRET-FLIM with the third partner. Four to five-week-old growth-chamber-grown Nicotiana benthamiana plants have been used as the model plant system for this demonstration.

Here we present, a method to visualize ternary complex formation between three protein partners using fluorescent-tagged proteins by BiFC based FRET-FLIM assay. This method is valuable for studying protein-protein interaction complexes in vivo.

Determinación de la interacción tripartita entre dos monómeros de un factor de transcripción MADS-box y una proteína sensora de calcio mediante ensayo BiFC-FRET-FLIM

Protein-protein interactions are an integral part of all biological processes in the cells as they play a crucial role in regulating, maintaining, and ...

Producción e imagen de vesículas recombinantes llenas de proteínas a partir de E. coli

Optimized Production and Analysis of Recombinant Protein-Filled Vesicles from E. coli

This innovative system, using a short peptide tag, that exports multiple recombinant proteins in membrane bound vesicles from E. coli, provides an effective solution to a range of problems associated with bacterial recombinant protein expression. These recombinant vesicles compartmentalise proteins within a micro-environment that facilitates the production of otherwise challenging, toxic, insoluble, or disulfide-bond containing proteins from bacteria. Protein yield is increased considerably when compared to typical bacterial expression in the absence of the vesicle-nucleating peptide tag. The release of vesicle-packaged proteins supports isolation from the culture medium&#160;and permits long-term active protein storage. This technology gives rise to increased yields of vesicle-packaged, functional proteins for simplified downstream processing for a diverse range of applications from applied biotechnology to discovery science and medicine. In the present article and the associated video, a detailed protocol of the method is provided, which highlights key steps in the methodology to maximize recombinant protein-filled vesicle production.

Autor destacado: La producción de proteínas recombinantes 

The present protocol describes a detailed method for the bacterial production of recombinant proteins, including typically insoluble or disulfide-bond containing proteins, packaged inside extracellular membrane-bound vesicles. This has the potential to be applied to versatile areas of scientific research, including applied biotechnology and medicine.