Name: probing high-density functional protein microarrays to detect protein-protein interactions
Uploaded: 2015-08-02T12:00:00.000+00:00
Duration: 8 min 7 s
Description: Watch this Scientific Journal Video about Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions at JoVE.com

This work was supported by a grant from the NIH. The assays shown in Figure 1 was performed by Dr. Joseph Fasolo. We thank Dr. Rui Chen for helpful comments.

1. Preparación de la sonda <ol><li> Cultura y purificar las sondas V5-quinasa de fusión utilizados para examinar las interacciones con otras proteínas como sigue: <ol><li> Utilice recién rayada cepa Y258 de levadura (MATa pep4-3, his4-580, ura3-53, leu2-3,112) que contiene proteína V5-fusión (expresado desde GATEWAY vector pYES-DEST52). Utilice la proteína aislada como sonda en los microarrays. Placa de la levadura en completa-uracilo sintético (Sc-Ura) / 2% de dextrosa / Agar y crecer a 30 ° C durante 3 días a partir de cultivo congelado (-80 ° C glicerol). </li><li> Inocular cultivos iniciadores (5-20 ml) de una sola colonia y crecer durante la noche en SC-ura / 2% de dextrosa en agitación plataforma (220-250 rpm) o rueda a 30 ° C. </li><li> A la mañana siguiente, inocular 400 ml de la Sc-Ura / 2% cultura rafinosa con cultivo iniciador suficiente para una DO final de 600 0.1. </li><li> Cultivar los inóculos a OD 600 de 0,6, seguido de galactosa inducción de la fu V5-quinasaSion constructo de expresión mediante la adición de una solución de levadura Extracto de 3x / peptona (YEP) suplementado con 6% de galactosa, mediante la adición de suficiente para diluir los medios de inducción por un factor de 3 para que concentración final de galactosa es 2%. </li><li> Inducir a las células a 30 ° C durante 6 horas en una plataforma de agitación. Utilice un matraz de 2 L Erlenmeyer para asegurar la aireación adecuada. </li><li> Cosecha células utilizando un rotor JA-10 (o comparable) haciendo girar 400 ml de suspensión celular a 1000 xg durante 5 min a 4 ° C. </li><li> Se lavan las células una vez con 50 ml de hielo búfer y transferir a un tubo cónico de 50 ml de PBS frío. Lavar el pellet de nuevo en hielo tampón PBS frío (sin detergentes u otros aditivos) que se utilizan para la lisis y traslado a 2 ml complemento tapa tubos para la lisis. </li><li> Girar las células a 20.000 xg durante 1 min a 4 ° C en un precipitado y pipeta de distancia el búfer. </li><li> Coloque los tubos en hielo y continúe con el paso de lisis. </li><li> Lyse las células (250-350 l pellet) con 00,5 mm perlas de óxido de circonio en una proporción 1: 1: 1 volumen de sedimento celular, cuentas, y solución salina tamponada con fosfato (PBS) lisis de amortiguación y agitar la mezcla usando una plataforma de agitación 3 veces a intervalos de 2 min a 4 ° C. </li><li> Centrifugar el lisado a 20.000 xg en una microcentrífuga de sobremesa durante 10 min a 4 ° C. </li><li> Pipeta sobrenadante en el tubo de centrífuga de policarbonato de alta velocidad y aclarar por ulracentrufugation durante 30 minutos a 150.000 xg a 4 ° C. </li><li> Transferir el lisado aclarado a un tubo que contiene resina de Ni 2+ prelavado afinidad (~ 100 l) y se incuba en un nutator durante 2 horas a 4 ° C para capturar histidina (His) 6X proteína marcada V5-fusión. </li><li> Lavar la resina tres veces durante 10 minutos a 4 ° C con tampón de lavado. Se precipitan la resina usando una centrífuga de mesa durante 5 min a 1000 xg a 4 ° C y aspirar el sobrenadante. Añadir tampón de lavado fresca para cada lavado y devolver el tubo de la nutator para la agitación durante el lavado. </li><li> Después del lavado steps son completos, aplicar la resina se lava a una columna fresca G-25 para la elución. </li><li> Aplicar 25 l de tampón de elución de una manera paso a paso empezando con 100 mM de imidazol a 500 mM de imidazol en incrementos de 50 mm para un total de 9 fracciones. </li><li> Ensayo de fracciones recogidas usando gel de poliacrilamida dodecil sulfato sódico (SDS-PAGE) y tinción de Coomassie para identificar la fracción (s) más altamente enriquecido para uso en el ensayo y añadir glicerol al 30% y se almacena a -80 ° C hasta su uso en el ensayo. </li></ol></li></ol> 2. Sondeo los Arrays NOTA: Por favor consulte el paso 2.6 de esta sección para preparar la solución de anticuerpos antes de comenzar el ensayo. <ol><li> Para detectar interacciones, diluir el anticuerpo conjugado-V5 fluoróforo (es decir AlexaFluor 647) a 260 ng / ml en tampón sonda y mezclar bien por agitación. NOTA: Preparar esta solución de anticuerpo 30 min antes de su uso y colocar el tubo en un nutato o rueda para asegurar una mezcla completa y una suspensión homogénea de anticuerpos. </li><li> Diluir la sonda proteína V5-fusión en un rango de concentración de 5-500 g / ml. NOTA: Optimizado para cada ensayo interacción proteína-proteína, utilizando tampón sonda. Optimización implica la adición de más sondas para sondear tampón. Típicamente 10 mg / ml se utiliza como punto de partida y ajusta en consecuencia sobre la base de la intensidad de la señal. </li><li> Retire los microarrays de proteínas del congelador (-20 ° C) y llevar a 4 ° C en el refrigerador justo antes de su uso. </li><li> Añadir tampón de bloqueo directamente al soporte de diapositivas que contiene los micorarrays proteínas y cubrir la parte superior con parafilm para evitar fugas. Bloquear las matrices en tampón de bloqueo durante 1 hr por agitación a 50 rpm en un escenario a 4 ° C. </li><li> Después del bloqueo, transferir los arrays a una cámara humidificada enfriado a 4 ° C, y añadir 90 l de sonda diluida directamente a la superficie de la matriz. Superposición del arrays con un resbalón elevador levantado e incubar estática (sin agitación) en la cámara húmeda a 4 ° C durante 1,5 horas. </li><li> Lavar las matrices 3 veces durante 1 min cada uno en tampón de sonda en tres tubos de 50 ml cónicos. Añadir la diapositiva para tubos cónicos que contienen suficiente búfer sonda pre-enfriado para envolver completamente el portaobjetos. Deje que el deslizamiento elevador se deslice suavemente fuera de los microarrays de proteínas (no lo fuerce fuera ya que esto podría causar daños a la superficie de la matriz). </li><li> Aplicar la solución de anticuerpos directamente a la matriz inmediatamente después de completar el lavado (paso 2.5) y la superposición con una hoja de levantador planteado como antes. Incubar las matrices de 30 min a 4 ° C en la cámara de humidificado. </li><li> Realice el mismo paso de lavado como antes (3 veces 1 min en tampón de sonda), y centrifugado en un tubo cónico de 50 ml a 800 xg en una centrífuga de mesa durante 5 min a temperatura ambiente. Seque las matrices en un soporte de diapositivas en la oscuridad durante 30 minutos antes de la exploración de la matriz en 647 nm. </li></ol>

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The authors declare that they have no competing financial interests.

El protocolo presentado se realizó originalmente con 85 proteínas únicas fusión quinasa-V5 de proteínas de levadura para comparar la actividad de unión a través de familias distintas y relacionadas de quinasas de proteínas de levadura que resulta en la identificación de nuevas redes de interacción quinasa in vivo 1. Como la tecnología de perfiles proteómicos emergentes, el desarrollo de alto rendimiento (HTP) proyección de proteomas utilizando microarrays de proteínas confiado en biblio...

La necesidad de realizar análisis globales de bioquímica de proteínas y la actividad in vivo de unión se ha traducido en el desarrollo de nuevos métodos para perfiles de interacciones proteína-proteína (IBP) y las modificaciones post-traduccionales de proteomas enteros 1,3-8. Microarrays de proteínas se fabrican como los microarrays de proteínas funcionales utilizando proteínas funcionales de larga duración 4-6,8,9 o microarrays de proteínas que contienen anticuerpos analíticos 10,11. Están diseñados para contener una alta densidad de proteínas dispuestas en portaobjetos de microscopio con una variedad de químicas de superficie para facilitar una variedad de condiciones experimentales requeridas para la realización de bioquímica amplio análisis 12. Nitrocelulosa y de superficie aldehído químicas para la unión química a través de métodos de fijación de lisina o de afinidad tales como toboganes quelatos de níquel para la fijación de proteínas etiquetadas-Su y el glutatión para la fijación de afinidad entre otros 13. </p&gt; El uso de microarrays de proteínas funcionales para detectar interacciones proteína-proteína requiere acceso a una biblioteca de alta calidad de proteína funcional 14. S. cerevisiae es susceptible de producir tal una biblioteca a través de la vinculación de la afinidad alta copia etiquetados construcciones de proteínas con alto rendimiento técnicas de purificación cromatográfica. La gran mayoría de genoma de la levadura ha sido secuenciado y casi todo el proteoma puede ser expresada a partir de un plásmido de alta copia para la purificación y análisis bioquímicos 12. Una vez que se obtienen las proteínas y dispuestas en formato de 384 pocillos, que se imprimen en un portaobjetos de microscopio que permite un rápido análisis bioquímico multi-paramétrico paralelo e interrogatorio bioinformático 8,14-16. Microarrays de proteínas se han utilizado para ensayos enzimáticos y de las interacciones con proteínas, lípidos, moléculas pequeñas, y ácidos nucleicos, entre muchas otras aplicaciones. La accesibilidad de las proteínas en la superficie del proteomaarrays los hacen susceptibles a diferentes tipos de detección analítica, incluyendo inmune afinidad, resonancia de plasmón superficial, la fluorescencia y muchas otras técnicas. Además, permite un control preciso de la condición experimental donde podría ser difícil de hacer en vivo. El objetivo de este protocolo es demostrar el uso apropiado de los microarrays de proteínas funcionales para detectar interacciones proteína-proteína. Esta aplicación permite el análisis bioquímico paralelo de alto rendimiento de las actividades de unión a proteínas utilizando un analito altamente purificada (proteína) de interés. Un C-terminal (carboxi-terminal) etiquetada proteína cebo V5-fusión de interés se produce a partir de un plásmido de alta copia en una cepa de levadura optimizado para la purificación de proteínas. Marcado C-terminal asegura que la proteína de longitud completa se ha traducido. La proteína utilizada en este estudio es Tda1-V5 proteína de fusión de cinasa, que se purificó usando resina de afinidad de níquel a través de una etiqueta His6X. La construc fusión Tda1-V5t se purifica a través de elución serial usando un gradiente de imidazol para eluir la fracción más altamente enriquecido para su uso en el ensayo.

<table><tbody><tr><td>Petri Dishes</td><td>VWR</td><td>NC-10747</td><td>or comparable</td></tr><tr><td>Bacto yeast extract</td><td>Difco</td><td>0217-17</td><td></td></tr><tr><td>Bacto peptone</td><td>Difco</td><td>0118-17</td><td></td></tr><tr><td>Bacto agar</td><td>Difco</td><td>0140-01</td><td></td></tr><tr><td>Dextrose</td><td>Sigma</td><td>D9434</td><td></td></tr><tr><td>Raffinose</td><td>Sigma</td><td>R0514</td><td></td></tr><tr><td>Galactose</td><td>Sigma</td><td>G0750</td><td></td></tr><tr><td>Sc-Ura drop out media</td><td>MP Bio</td><td>114410622</td><td></td></tr><tr><td>Small Culture tubes</td><td>VWR/Fisher</td><td></td><td></td></tr><tr><td>Large Erlenmeyer Flask</td><td>VWR/Fisher</td><td></td><td></td></tr><tr><td>Centrifuge JA-10 rotor</td><td></td><td></td><td></td></tr><tr><td>Centrifuge tubes (500 ml)</td><td></td><td></td><td></td></tr><tr><td>Falcon tube (50 ml)</td><td>VWR/Fisher</td><td>21008-951</td><td></td></tr><tr><td>PBS Tablets</td><td>Sigma</td><td>P4417</td><td></td></tr><tr><td>FastPrep Tubes (2 ml screw cap tube)</td><td></td><td></td><td></td></tr><tr><td>FastPrep Machine</td><td>MP Biomedicals</td><td>116004500</td><td></td></tr><tr><td>Zircon Beads</td><td>BioSpec</td><td>11079105</td><td></td></tr><tr><td>Triton X100</td><td>Sigma</td><td>P4417</td><td></td></tr><tr><td>DTT</td><td>Sigma</td><td>D9779</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M8266&amp;nbsp;</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S3014&amp;nbsp;</td><td></td></tr><tr><td>Imidazole (pH = 7.4)</td><td>Sigma</td><td>I5513&amp;nbsp;</td><td></td></tr><tr><td>PMSF</td><td>Sigma</td><td>93482</td><td></td></tr><tr><td>Complete Inhibitor Cocktail (EDTA Free)</td><td>Roche</td><td>11873580001</td><td></td></tr><tr><td>Phosphatase inhibitor cocktail 1</td><td>sigma</td><td>P2850</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A2153&amp;nbsp;</td><td></td></tr><tr><td>Tween-20</td><td>Sigma</td><td>P1379&amp;nbsp;</td><td></td></tr><tr><td>ATP</td><td>Sigma</td><td>A1852&amp;nbsp;</td><td></td></tr><tr><td>Shaking Incubator (30 &amp;deg;C)</td><td></td><td></td><td></td></tr><tr><td>Nickel Affinity Resin</td><td>Life Technologies</td><td>R901-01</td><td></td></tr><tr><td>G25 column</td><td>GE life sciences</td><td>27-5325-01&amp;nbsp;</td><td></td></tr><tr><td>Nutator</td><td>VWR/Fisher</td><td></td><td></td></tr><tr><td>SDS-PAGE Gel (NuPage)</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Coomassie Blue Stain</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Monoclonal V5 Antibody</td><td>Life Technologies</td><td>R960-25</td><td></td></tr><tr><td>Alexa647 Tagged monoclonal V5 Antibody</td><td>Life Technologies</td><td>451098</td><td></td></tr><tr><td>Protein Microarrays Kit</td><td>Life Technologies</td><td>PAH0525013</td><td></td></tr><tr><td>Genepix Scanner</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Lifter Slips</td><td>Thomas Scientific</td><td></td><td>http://www.thomassci.com/Supplies/Microscope-Cover-Glass/_/LIFTER-SLIPS/</td></tr><tr><td>Lysis Buffer: 0.1% Triton X-100, 0.5 mM DTT,&lt;br/&gt; 2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 500 mM NaCl, 50 mM imidazole (pH 7.4), Complete protease inhibitor cocktail &amp;ndash; EDTA-free, Phosphatase inhibitor Cocktail 1, 1 mM PMSF&amp;nbsp; (add just prior to use)</td><td></td><td></td><td>Add the reagents to 1x PBS (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride; pH 7.4) to obtain the desired volume of lysis solution</td></tr><tr><td>Blocking Buffer: 1% bovine serum albumin (BSA), 0.1% Tween-20</td><td></td><td></td><td>Add the reagents to 1x PBS to obtain the desired volume of blocking buffer</td></tr><tr><td>Probe Buffer: 2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 0.5 mM DTT, 0.05% Triton X-100, 50 mM NaCl, 500 &amp;mu;M ATP (for kinases), 1% BSA</td><td></td><td></td><td>Add the reagent to 1x PBS to obtain desired volume of probe buffer</td></tr><tr><td>Wash Buffer: 0.1% Triton X-100, 500 mM NaCl, 0.5 mM DTT, 50 mM imidazole (pH 7.4),2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td>Add the reagent to 1x PBS to obtain desired volume of probe buffer</td></tr><tr><td>Elution Buffer: 0.1% Triton X-100, 0.5 mM DTT, 2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 500 mM NaCl, 50 &amp;ndash; 500 mM imidazole (pH 7.4), Complete protease inhibitor cocktail &amp;ndash; EDTA-free, Phosphatase inhibitor Cocktail 1, 1 mM PMSF (add just prior to use)</td><td></td><td></td><td>Before you begin, it is important to note the concentration gradient of imidazole (50 &amp;ndash; 500 mM) and to create separate tubes for each concentration (it is advisable to create the interval using 50 mM increments). Add the reagent to 1x PBS to obtain desired volume of probe buffer</td></tr><tr><td>3x YEP +6% galactose: Dissolve 30 g of yeast extract and&amp;nbsp; 60 g of peptone in 700 ml of H2O, and autoclave. Add 300 ml of filter sterilized 20% galactose (wt/vol)</td><td></td><td></td><td>&amp;nbsp;Galactose should not be autoclaved</td></tr></tbody></table>

probing high-density functional protein microarrays to detect protein-protein interactions

De alta densidad de microarrays de proteínas funcionales que contienen ~ 4200 proteínas de levadura recombinantes se examinan las interacciones proteína-proteína quinasa utilizando una proteína de fusión purificada por afinidad levadura quinasa que contiene una etiqueta V5-epítopo para leer-out. Quinasa purificada se obtiene mediante cultivo de una cepa de levadura optimizado para la producción de proteínas de copia alto que alberga un plásmido que contiene una fusión de quinasa-V5 construir bajo un promotor inducible GAL. La levadura se cultiva en medios de comunicación restrictiva con una fuente de carbono neutral durante 6 horas seguido de inducción con 2% de galactosa. A continuación, el cultivo se recoge y se purifica usando quinasa de afinidad estándar técnicas cromatográficas para obtener una proteína quinasa altamente purificada para su uso en el ensayo. La quinasa purificada se diluye con tampón de quinasa a un rango apropiado para el ensayo y los microarrays de proteínas están bloqueados antes de la hibridación con el microarray de proteínas. Después de la hibridación, los arreglos se probaron con antib V5 monoclonalody para identificar proteínas unidas por la proteína quinasa-V5. Por último, las matrices se escanean con un escáner de microarrays estándar, y los datos se extrajeron para el análisis de la informática aguas abajo 1,2 para determinar una alta confianza conjunto de interacciones de proteínas para la validación de aguas abajo en vivo.

Se observó la actividad de interacción proteína-proteína usando un lector de chip estándar para evaluar la fusión de la proteína quinasa Tda1-V5 constructo como una proteína de cebo contra una levadura de microarrays proteína funcional que contiene aproximadamente 4.200 única S. las proteínas de fusión GST cerevisiae. Además de interrogación con el software Genepix reveló una multitud de eventos de diferentes intensidades de unión. La afinidad se mide a partir de la intensidad graduada ...

Watch this Scientific Journal Video about Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions at JoVE.com

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

El uso de microarrays de proteínas que contienen casi toda la S. cerevisiae proteoma se sondeó para un rápido interrogatorio imparcial de miles de interacciones proteína-proteína en paralelo. Este método puede ser utilizado para la molécula de proteína pequeña, modificación postraduccional, y otros ensayos en alto rendimiento.

Sondeo de alta densidad microarrays de proteínas funcionales para detectar interacciones proteína-proteína

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

probing-high-density-functional-protein-microarrays-to-detect-protein

Research

JoVE Journal

Biology

8.0K vistas.  Stanford University.  El uso de microarrays de proteínas que contienen casi toda la S. cerevisiae proteoma se sondeó para un rápido interrogatorio imparcial de miles de interacciones proteína-proteína en paralelo. Este método puede ser utilizado para la molécula de proteína pequeña, modificación postraduccional, y otros ensayos en alto rendimiento. 

Video: Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

Tecnología de microarrays de proteínas extracelulares para detección de alto rendimiento de las interacciones ligando-Receptor de baja afinidad

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Investigación

Bioquímica

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

Un enfoque comparativo para caracterizar el paisaje del Huésped-Patógeno interacciones proteína-proteína

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Inmunología e Infección

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

En vivo-La detección de interacciones proteína-proteína en la Micro-patrón superficies

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Biología

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

Anticuerpos, químicamente bloqueado Microarray de multiplexado de alto rendimiento de perfiles de glicosilación de proteínas específicas en muestras complejas

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous proteomes, including those from viruses, bacteria, and yeast.&#160; In comparison, analysis of the human proteome lags behind, partially due to the sheer number of proteins which must be studied, but also the complexity of networks and interactions these present. To specifically address the challenges of understanding the human proteome, we have developed HaloTag technology for protein isolation, particularly strong for isolation of multiprotein complexes and allowing more efficient capture of weak or transient interactions and/or proteins in low abundance. &#160;HaloTag is a genetically encoded protein fusion tag, designed for covalent, specific, and rapid immobilization or labelling of proteins with various ligands. Leveraging these properties, numerous applications for mammalian cells were developed to characterize protein function and here we present methodologies including: protein pull-downs used for discovery of novel interactions or functional assays, and cellular localization. We find significant advantages in the speed, specificity, and covalent capture of fusion proteins to surfaces for proteomic analysis as compared to other traditional non-covalent approaches. We demonstrate these and the broad utility of the technology using two important epigenetic proteins as examples, the human bromodomain protein BRD4, and histone deacetylase HDAC1.&#160; These examples demonstrate the power of this technology in enabling &#160;the discovery of novel interactions and characterizing cellular localization in eukaryotes, which will together further understanding of human functional proteomics. &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;

HaloTag technology is a multifunctional technology which has shown significant success in isolation of both small and large protein complexes from mammalian cells.&#160; Here we highlight the advantages of this technology compared to existing alternatives and demonstrate its utility to study numerous aspects of protein function inside eukaryotic cells.

El descubrimiento de las interacciones de proteínas y caracterización de la función de proteínas utilizando HaloTag Tecnología

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous ...

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 ...

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 ...

Detección de alta sensibilidad de la unión de proteínas compuestas mediante fluorescencia

Detection of CD40 Protein-Umbelliferone Interaction via Differential Scanning Fluorescence

The investigation of interactions between different molecules is a crucial aspect of understanding disease pathogenesis and screening for drug targets. Umbelliferone, an active ingredient in Tibetan medicine Vicatia thibetica, exhibits an immunomodulatory effect with an unknown mechanism. The CD40 protein is a key target in the immune response. Therefore, this study employs the principle of differential scanning fluorescence technology to analyze the interactions between CD40 protein and umbelliferone using fluorescent enzyme markers. Initially, the stability of the protein fluorescent orange dye was experimentally verified, and the optimal dilution ratio of 1:500 was determined. Subsequently, it was observed that the temperature melting (Tm) value of CD40 protein tended to decrease with an increase in concentration. Interestingly, the interaction between CD40 protein and umbelliferone was found to enhance the thermal stability of CD40 protein. This study represents the first attempt to detect the binding potential of small molecule compounds and proteins using fluorescence microplates and fluorescent dyes. The technique is characterized by high sensitivity and accuracy, promising advancements in the fields of protein stability, protein structure, and protein-ligand interactions, thus facilitating further research and exploration.

Autor destacado: Optimización de los métodos de PCR para mejorar la accesibilidad y la eficiencia

This protocol describes a unique method for detecting the binding between compounds and protein molecules, offering the advantages of minimal protein sample loss and high data accuracy.

Detección de la interacción proteína-umbeliferona CD40 mediante fluorescencia diferencial de barrido

The investigation of interactions between different molecules is a crucial aspect of understanding disease pathogenesis and screening for drug targets. ...

Sondeo de alta densidad microarrays de proteínas funcionales para detectar interacciones proteína-proteína

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