Agradecemos EUROSARF por enviarnos plásmidos de expresión de TAP levadura. También estamos agradecidos por la ayuda editorial de Ryan Labadens. Este trabajo fue apoyado por una beca NIH / NINDS (R01NS070962) a CW

1. Generar UAS-TAP-etiquetados moscas transgénicas <ol><li> Generar pUAST-TAP-etiquetados construcciones de ADN. <ol><li> Decidir qué lado (N-o C-terminal) de la proteína cebo la etiqueta grifo deberá estar fusionada a, basada en la estructura / función de la proteína. Véase la discusión para más detalles. </li><li> Subclónese la región de codificación de cDNA del gen de interés en los sitios de clonación múltiple (MCS) de los vectores pUAST-NTAP o pUAST-CTAP para generar N-o C-terminal de etiquetado transgenes UAS-TAP, respectivamente. Consulte la Figura 1 para los mapas detallados y sitios de restricción utilizables y marcos de lectura. </li></ol></li><li> Generar UAS-TAP-etiquetados moscas transgénicas. <ol><li> Generar moscas transgénicas siguiendo protocolos estándar utilizando P-elemento de inserción mediada por 11. Un número de servicios de inyección se encuentran disponibles comercialmente. </li><li> Cruce GAL4 conductor neuronal (es decir BG380-GAL4) a cada línea transgénica individual y determinar los niveles de expresión de proteínasde cada línea por transferencia de western (con anticuerpo anti-peroxidasa de peroxidasa) y / o la inmunotinción (con anticuerpo anti-TAP). En general, se recomienda una línea transgénica con un nivel de expresión de la proteína que se encuentra cerca de la proteína endógena para los procedimientos de TAP. Véase la discusión para más detalles. </li><li> PerformGAL4/UAS-based experimentos de rescate para confirmar la funcionalidad de los transgenes TAP-etiquetados si la pérdida de función de los mutantes de los genes de interés están disponibles. Elija un transgén que puede rescatar a la veracidad de los fenotipos mutantes para los siguientes experimentos TAP. </li></ol></li></ol> 2. Preparar muestras de Procedimiento TAP <ol><li> Generar una acción mosca que transmite tanto un controlador neuronal GAL4 (por ejemplo, BG380-Gal4) y el transgén de TAP-etiquetados elegido con el fin de facilitar la expansión de las muestras de la mosca. Recoger las progenies F1 de la GAL4 conductor y la cruz-UAS transgenes en los casos raros en que la combinación anterior hace que la supervivencia y el crecimiento de desventaja. &lt;/ Li&gt; <li> Recoger muestras a pequeña escala y optimizar condiciones lisis para solubilizar la proteína TAP-etiquetados. <ol><li> Hacer una serie de tampones de lisis utilizando una combinación de los detergentes no iónicos NP-40 (0,1-1%), NaDOC (0,1-1%) y Triton X-100 (0,05-0,5%). Véase la Tabla 1 y la discusión para más información. </li><li> En la parte superior de una almohadilla de CO2, utilice # 5 pinzas para diseccionar 20 cabezas adultas desde el transgén que expresa TAP moscas y recoger en un tubo de 1,5 ml para probar cada condición de tampón de lisis. </li><li> Añadir 100 ul de tampón de lisis para las pruebas del tubo y homogeneizar las cabezas acariciando arriba y abajo con una mano de mortero de plástico, a continuación, añadir otro búfer pruebas de 100 ul. </li><li> Girar la cabeza lisado en 21.500 xg durante 10 min (4 ° C), y separar el sobrenadante y el sedimento después de la centrifugación. Añadir 25 ul de tampón de carga 2x SDS al sedimento y tampón de carga de 10 ul 4x SDS a 30 ul de sobrenadante respectivamente. </li><li> Hervir las dos muestras durante 5 min y analizarlos lado a-Lado utilizando SDS-PAGE y posterior transferencia de Western con el anticuerpo PAP. Determinar la solubilidad por la relación de los niveles de proteína PAT en sobrenadante frente a la pastilla. </li></ol></li><li> Preparar la muestra en gran escala <ol><li> Expandir y recoger las moscas adultas. <ol><li> Ampliar las acciones neuronal-GAL4/UAS-TAP-transgene en botellas y voltear las botellas cada 3 días hasta 250 botellas acumulativos se utilizan para la recolección. </li><li> Recoger 1-3 días de edad moscas adultas en 50 ml tubos cónicos, coloque el tubo en nitrógeno líquido inmediatamente a lo profundo del congelamiento de las moscas. Guarde las moscas en un -80 ° C congelador. Tenga en cuenta que el volumen de las moscas no debe superar los 2/3 del tubo de 50 ml. </li></ol></li><li> Recoger las cabezas de mosca (realizar este paso en la parte superior del hielo seco en polvo). <ol><li> Saque los tamices preenfriado y el mortero y la maja de la -80 ° C congelador y ponerlos en hielo seco, idealmente dentro de un gran cubo de hielo. Apila dos tamices de ensayo estándar USA con un número 25 en el the superior y N º 40 en la parte inferior. </li><li> Tome las moscas congeladas y soltarlos en nitrógeno líquido y mantener las moscas allí por unos 10 minutos. Vortex o agitar los tubos vigorosamente para romper las cabezas, piernas y alas de los cuerpos. </li><li> Vierta la mezcla en el tamiz superior, y luego sacudir los tamices vigorosamente mientras sujeta ambos tamices juntos. Después de cribado, los cuerpos se quedan en el tamiz superior, volar cabezas serán retenidos en el tamiz de fondo, y las piernas, alas, y otros escombros caerán al hielo seco. Separar los dos tamices y cuidadosamente transferir las cabezas de mosca al mortero frío. </li></ol></li><li> Homogeneizar las cabezas de mosca <ol><li> En la parte superior del hielo seco, moler las cabezas con el mortero y la mano del mortero a las partículas en polvo, y luego transferir el polvo a una de 15 ml de vidrio Dounce triturador de tejidos que se preenfriar en hielo. </li><li> Medir el peso de la amoladora antes y después de la muestra de la cabeza se vertió en él, y luego calcular cuánto samp cabezale pesa. Un total de 6-15 gramos de cabezas de moscas será suficiente para cada experimento TAP ajustar en consecuencia a los niveles de expresión de la proteína. Añadir 15 ml de tampón de homogeneización enfriado con hielo (tampón de lisis optimizado en el paso 2.2) para el polvo y luego golpe con la mano de mortero de gran holgura hasta que sea fácil para los maja que suben y bajan. Mantenga el triturador de vidrio en el hielo en todo momento. </li></ol></li><li> Preparar el sobrenadante de TAP <ol><li> Transferir el homogeneizado a un tubo de centrífuga de alta velocidad y efecto durante 20 minutos a 50.000 xg ~ (4 ° C). Transferir el sobrenadante a un nuevo tubo de centrífuga de alta velocidad y repetir la centrifugación una vez más. </li><li> Transferir el sobrenadante a un tubo de ultracentrífuga y realizar una 40 min ~ 250.000 xg de centrifugado para eliminar aún más el sobrenadante. El sobrenadante está listo para los procedimientos de purificación por afinidad en tándem después de ultracentrifugación. </li></ol></li></ol></li></ol> 3. Purificación TAP </p&gt; En las siguientes secciones se derivaron de la TAP laboratorio Séraphin protocol 12 (http://web.as.uky.edu/Biology/faculty/rymond/BIO%20510/Bertran%20Seraphin%27s%20TAP%20page.pdf ) <ol><li> Realice IgG grano purificación por afinidad <ol><li> Preparar cordón de sefarosa de IgG, mientras que las muestras se centrifugaron. Lave 400 l 3x perlas de IgG en un tubo Falcon de 15 ml con 10 ml de tampón de lavado frío IgG. Para cada lavado, sacudir el tubo suavemente durante 2 minutos, y luego de girar las bolas a 1.000 xg durante 2 min. Al final del tercer lavado, eliminar el tampón y dejar sólo las perlas en el tubo. </li><li> Transferir con cuidado el sobrenadante clarificado (~ 15 ml) en el tubo de 15 ml que contiene las perlas de IgG. Incubar las cuentas y mezcla lisado cerebral a 4 º C en un nutator durante 2 horas. </li><li> Configurar una columna micro limpio y vacío con aproximadamente 15 ml de volumen total en el cuarto frío. Cargue la mezcla de glóbulos IgG vertiendo constantemente la mezcla en la columna, no trate de atrapar a cualquierburbujas de aire en el interior de la columna. Permitir que las perlas se asientan en la columna y el tampón para drenar lentamente por gravedad. </li><li> Lavar la columna a fondo con 10 ml de tampón de lavado IgG fría después de todo el lisado cerebral ha fluido a través de la columna de IgG resuelto. Repita el lavado 2x. Nota: nunca deje que el cordón seco en el aire. </li></ol></li><li> Realice TEV división <ol><li> Después del tercer lavado, lavar la columna de nuevo con 10 ml de tampón de disociación TEV. Este paso se prepara el cordón de IgG que secuestra el complejo de cebo para TEV división. </li><li> Justo antes de la última gota de tampón de TEV está a punto de gotear, poner un tapón en la parte inferior de la columna para bloquear el flujo, añadir 1,3 ml de tampón de TEV que contiene 130 unidades de enzima TEV a la columna, y a continuación, tape de forma segura la parte superior de la columna. Asegúrese de que la columna se sella bien en ambos extremos. </li><li> Gire la columna a 18 º C durante 2 horas para permitir que la enzima TEV para escindir el péptido en el lugar de TEV y suelte el WHI complejo proteicole dejando atrás la proteína Un péptido de dominio vinculado a las perlas de la IgG sepharose. </li></ol></li><li> Realice Calmodulina grano purificación por afinidad <ol><li> Preparar las perlas Calmodulin mientras que las perlas de IgG se incubaron con la enzima de TEV. Lave 200 l perlas Calmodulin en unos 15 ml 3x tubo Falcon, cada vez con 10 ml de frío Calmodulina de tampón de unión. Para cada lavado, agite con cuidado el tubo durante 2 minutos en un nutator, y luego de girar las bolas a 1.000 xg durante 2 min. Al final del tercer lavado, saque todo el buffer y dejar sólo las perlas en el tubo. </li><li> Al final de la incubación TEV (paso 3.2.3), devuelva la columna de la IgG de nuevo a la cámara frigorífica y déjelo a un derecho para arriba. Deje que los granos se conforman con 10 min. </li><li> Quite la tapa superior y luego la tapa inferior y, a continuación, recoger el producto de escisión ml TEV 1,3 en un tubo Falcon de 15 ml. Deje que el buffer se drene completamente. Añadir un tampón de 200 l de TEV adicional a la columna para empujar hacia fuera el volumen muerto de la columna, recoger el fbaja en el mismo tubo. </li><li> Añadir 4,5 ml de tampón de unión de calmodulina y 4,5 l 1 M de CaCl2 al producto de escisión 1,5 ml de TEV recogido anteriormente. El CaCl2 sirve para valorar el EDTA en el tampón de TEV. Transferir la mezcla de 6 ml al tubo que contenía las perlas de calmodulina y girar el tubo a 4 º C en un nutator durante 1 hora. </li><li> Crear otra columna micro limpio y vacío con aproximadamente 10 ml de volumen total en el cuarto frío. Cargar la mezcla de glóbulos de calmodulina a la columna y dejar que se drene por gravedad. </li><li> Cuando toda la solución ha fluido a través de la columna de calmodulina asentado, lavar la columna dos veces, cada una con 10 ml de calmodulina de tampón de unión frío. Nota: no molestar a los granos de calmodulina y tratar de mantener la superficie de las perlas tan planas como sea posible durante el lavado. </li></ol></li><li> Eluir el complejo de cebo de la columna de calmodulina. Inmediatamente después del lavado, eluir la columna de la calmodulina con cinco fracciones de 200 l frío Calmodutampón de elución Lin. Para cada fracción, agregar suavemente 200 l de tampón de elución a la columna y recoger el eluido con una marcada 1,5 ml tubo Eppendorf. Repita este 4x. </li><li> Analizar el complejo de proteínas por SDS-PAGE <ol><li> Tomar una pequeña alícuota de cada una de las cinco fracciones (unos 30 l) y añadir tampón de carga SDS. Hervir las muestras durante 5 min y la carga de las muestras de lado a lado con la proteína de marcadores moleculares en un gradiente (4-15%) en gel de SDS-PAGE. </li><li> Después de que las muestras han resuelto por completo en el gel, detener la electroforesis y teñir el gel con cualquier procedimiento coloidales sensibles basados-G-250 de Coomassie de tinción tales como &#39;plata azul&#39; tinción 13. La tinción con plata es opcional, pero no es preferible debido a que no es totalmente compatible con el análisis de espectrometría de masas posterior. Almacenar el resto del eluido en un congelador -80 ° C para su posterior análisis, tales como la espectrometría de masas para el descubrimiento de las identidades moleculares del complejo de proteína purificada. See discusión. </li></ol></li></ol>

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Método de purificación por afinidad en tándem (PAT) ofrece un protocolo de purificación dual que permite el aislamiento y enriquecimiento de complejos de proteínas a través de dos etapas de purificación de afinidad independientes. El diseño de la etiqueta TAP no se limita a lo que se presenta en este protocolo, otros dominios y motivos de unión de proteínas también son aplicables si las condiciones del tampón se ajustan en consecuencia. Un buen ejemplo de otras etiquetas de TAP es la etiqueta GS-TAP, una com...

Definición de las vías moleculares o redes que median un proceso biológico en particular es uno de los objetivos fundamentales de la investigación biomédica. Fly genetistas han dependido en gran medida de la genética a plazo, especialmente modificador pantallas genéticos (tanto potenciadoras y pantallas supresoras), para identificar los factores que trabajan juntos, en paralelo con, o aguas arriba o aguas abajo de un gen de interés. Sin embargo, las pantallas de genética hacia adelante muchas veces fallan en identificar los genes esenciales que, cuando muta, causa la letalidad en las etapas tempranas del desarrollo, o los genes con redundancia funcional y una indemnización, cuya pérdida de la función única causa defectos sutiles que son difíciles de marcar. Una forma de superar esta dificultad es la detección de interacciones directas proteína-proteína. Durante más de una década, una creciente lista de métodos bioquímicos, incluyendo la levadura de dos híbridos, visualización de fagos, entrecruzamiento químico, Co-IP, Tandem Affinity purificación (TAP), etc. se han utilizado para investigar proteínas-proteína interacciones. Cada uno de estos enfoques tiene su propio conjunto de fortalezas y debilidades en cuanto a sensibilidad y especificidad. Entre ellos, el método de TAP permite la detección de la interacción física bajo condiciones casi fisiológicas, conserva la especificidad y la consistencia y 2 incluye la capacidad de extender al análisis de alto rendimiento de 3,4. El método TAP fue desarrollado originalmente en levadura por colegas Rigautand 1. En este método, una proteína de interés se expresa con una etiqueta de TAP. La etiqueta TAP alberga dos dominios de unión por afinidad independientes: una proteína de un dominio que se une a IgG y un dominio de unión a calmodulina. Los dos dominios están separados por una TEV (Tobacco Etch Virus) sitio de escisión. Tal combinación permite dos rondas independientes de purificaciones de afinidad para reducir suficientemente enlaces no específicos y enriquecen enlaces específicos 1. Para este ejemplo, el método TAP es una muy poderosa method para identificar en las interacciones in vivo de una proteína dada, aunque la sobreexpresión de la proteína exógena puede hacer que sea más propensos a asociarse con proteínas que normalmente no complejo con su homólogo endógeno. Desde su desarrollo, el método TAP se ha aplicado en muchos otros sistemas, incluidos los sistemas celulares basados ​​en la cultura y otros 5,6 sistemas modelo in vivo en 6-9. Aquí se describe la adaptación del método TAP en Drosophila. En primer lugar, generar vectores pUAST-NTAP y pUAST-CTAP para facilitar la clonación y la fusión de la etiqueta TAP para el extremo N-o C-terminal del gen de interés. El TAP-etiquetados-UAS transgén se expresa entonces en el sistema nervioso bajo el control de un controlador de GAL4 neuronal 10. A continuación, se recogerá un gran número de cabezas de moscas adultas, que tienen un alto contenido de células neuronales y son fáciles de separar de otras partes del cuerpo después de la congelación sobre la base de las diferencias de tamaño. Las cabezas de los adultos se homogeneizan y se aclaró bcentrifugaciones secuenciales y, y el sobrenadante está sujeto a un procedimiento de punción se describe a continuación.

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			<td style="width:119px;">04-881-18&#160;</td>
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			<td height="42" style="height:42px;">U.S.A. standard test sieve No. 40</td>
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			<td height="21" style="height:21px;">&#160;Kontes Dounce Tissue Grinders 15 ml</td>
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identifying protein-protein interaction in drosophila adult heads by tandem affinity purification (tap)

Pantallas genéticos realizados con Drosophila melanogaster (mosca de la fruta) han hecho numerosos descubrimientos hito en el avance de las ciencias biológicas. Sin embargo, el uso de pantallas bioquímicos destinados a ampliar los conocimientos adquiridos en el análisis genético fue explorado recientemente. Aquí se describe un método para purificar el complejo de la proteína que se asocia con cualquier proteína de interés a partir de cabezas de moscas adultas. Este método toma ventaja del sistema de Drosophila GAL4/UAS para expresar una proteína cebo fusionado con una etiqueta tándem Purificación por Afinidad (TAP) en las neuronas de la mosca in vivo, y luego implementa dos rondas de purificación utilizando un procedimiento de punción similar a la establecida originalmente en de levadura 1 para purificar el complejo de proteína de interacción. Al final de este procedimiento, se obtiene una mezcla de varios complejos de proteínas cuyas identidades molecular se puede determinar por espectrometría de masas. Validación de las proteínas candidatas se beneficiará de la resource y la facilidad de la realización de estudios de pérdida de función en las moscas. Enfoques similares se pueden aplicar a otros tejidos de la mosca. Creemos que la combinación de la manipulación genética y este enfoque proteómico en el sistema modelo de la mosca tiene un tremendo potencial para abordar los problemas fundamentales en el campo de la neurobiología y más allá.

Aquí se demuestra nuestro esfuerzo en la identificación de proteínas Highwire interaccionan en el cerebro de mosca. Highwire (Hiw) y sus homólogos de vertebrados e invertebrados son enormes ubiquitina ligasas que regulan el desarrollo y la reparación del sistema nervioso 14. Ellos comparten una serie de dominios funcionales altamente conservadas. Sin embargo, sus acciones moleculares no están del todo claras. El trabajo realizado en gusano, mosca y el ratón condujo al modelo de trabajo actual que HIW f...

Watch this Scientific Journal Video about Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP at JoVE.com

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila es famoso por su gran alcance de la manipulación genética, pero no por su idoneidad del análisis bioquímico en profundidad. Aquí presentamos un procedimiento basado en TAP para identificar socios que interactúan de cualquier proteína de interés a partir de cerebro de la mosca. Este procedimiento puede potencialmente conducir a nuevas vías de investigación.

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. ...

identifying-protein-protein-interaction-drosophila-adult-heads-tandem

Research

JoVE Journal

Biology

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

13.8K vistas.  Louisiana State University Health Sciences Center.  Drosophila es famoso por su gran alcance de la manipulación genética, pero no por su idoneidad del análisis bioquímico en profundidad. Aquí presentamos un procedimiento basado en TAP para identificar socios que interactúan de cualquier proteína de interés a partir de cerebro de la mosca. Este procedimiento puede potencialmente conducir a nuevas vías de investigación. 

Video: Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

Aislamiento y purificación de Drosophila Clasificación de las neuronas periféricas por bolas magnéticas

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Investigación

Biología

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

Purificación de proteínas ortogonales Facilitado por un marcador de afinidad biespecíficos pequeños

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

Identificación de los socios interacción de proteínas en tándem de purificación de afinidad

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Aislamiento de la cromatina mediante la purificación de ARN (chirp)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

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 ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Identificación de los complejos de proteínas en<em&gt; Escherichia coli</em&gt; La purificación mediante afinidad del péptido secuencial en combinación con espectrometría de masas tándem

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

Purificación de las transcripciones y los metabolitos de<em&gt; Drosophila</em&gt; Cabezales

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

Purificación de proteínas libre de Método de Determinación afinidad de unión por termoforesis Microscale

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

En Vivo Tracking 4-Dimensional de madre hematopoyéticas y células progenitoras en ratones adultos de calota de Médula Ósea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

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 ...

La identificación de la interacción proteína-proteína en

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