Los autores agradecen al personal del departamento de Biotecnología Farmacéutica y Biología Molecular y sobre todo Pelsmacker Monique De la preparación del virus de la etiqueta radiactivo. Estamos muy agradecidos a Elena de Merckx y Hadewych Halewyck por sus interesantes comentarios y discusiones y Gerrit De Bleeser por su ayuda en el laboratorio. Este trabajo fue apoyado financieramente por un subsidio OZR de la Vrije Universiteit Brussel (OZR1807), el Fonds voor Wetenschappelijk Onderzoek Vlaanderen (G.0168.10N) y la Organización Mundial de la Salud (TSA 200 410 791). Lise Schotte es un becario predoctoral del Fonds voor Wetenschappelijk Onderzoek Vlaanderen (FWO).

El principio (A) y una visión general del método (B) se muestra en la Figura 1. 1. Preparación del tampón <ol><li> Preparar Encuadernación / tampón de lavado por disolución de dihidrógeno fosfato sódico (50 mM) y cloruro de sodio (300 mM) en agua y ajustar el pH a 8,0. Adicionalmente añadir Tween 20 (0,01% (m / v) de concentración final), metionina (2% (m / v) de concentración final) y la albúmina (0,1% (m / v) de concentración final) y ajustar el volumen requerido. </li></ol> 2. Preparación de las perlas magnéticas Preparar las partículas magnéticas de acuerdo con el manual de instrucciones. En pocas palabras: <ol><li> Resuspender las perlas magnéticas usando un vórtice. </li><li> Transferencia, para cada muestra, 10 l de las perlas magnéticas resuspendidos (40 mg / ml) en un tubo. </li><li> Recoge las bolas magnéticas con un imán, hasta que el sobrenadante es claro (+ / - 30 segundos) y separar el sobrenadante con cuidado. </li><li> Lave elperlas magnéticas dos veces: volver a suspender las bolas en 150 l de unión / tampón de lavado. Migración de las perlas magnéticas a un lado del tubo utilizando un imán hasta que el sobrenadante es claro y quitar cuidadosamente el sobrenadante. </li><li> Utilizar 40 l de unión / tampón de lavado, para cada muestra, para volver a suspender las perlas (10 mg / ml). </li></ol> 3. Afinidad ensayo de captura Nota: Para medir la radiactividad de fondo (cósmico) (0% de radioactividad), ningún virus radiomarcado se añade a una muestra de control 1. En cambio, el mismo volumen Encuadernación / tampón de lavado, se añade. Nota: 100% radiactividad se define por una muestra de control 2 para que todos los componentes se añaden excepto el nanocuerpos. En cambio, el mismo volumen Encuadernación / tampón de lavado, se añade. <ol><li> Llevar 2000 cpm de 35 S etiquetados (β-radiación) poliovirus Sabin tipo de cepa 1 9, se diluyó con Encuadernación / tampón de lavado a 80 l en una placa de microtitulación de 96 pocillos. </li><li> Añadir 10 &amp; mu, l de dilución nanocuerpos a los pocillos. </li><li> Mezclar durante unos 10 segundos con un agitador. </li><li> Permitir que las muestras se incuban durante 1 hora a temperatura ambiente. </li><li> Añadir 40 l de la suspensión lavada perlas magnéticas y se incuba durante 10 minutos a temperatura ambiente, bajo agitación continua. </li><li> Separar las perlas y el sobrenadante utilizando un imán y transferir el sobrenadante aclarado en un tubo. </li></ol> 4. La medición de la radiactividad <ol><li> Transferir 50 l del sobrenadante de la etapa 3.6 en un matraz de escrutinio. </li><li> Añadir 3 ml de líquido de centelleo y se mezcla. Medir la radiactividad en un contador de centelleo β-. </li></ol> 5. El reciclaje de las perlas <ol><li> Recoger las perlas magnéticas utilizadas en un tubo y se centrifuga a 500 xg durante 2 minutos o hasta que un sobrenadante claro se obtiene. </li><li> Eliminar el sobrenadante y resuspender las cuentas en 6 ml de NaOH 0,5 M. </li><li> Transferir la suspensión a multipltubos electrónicos y se incuba en un baño de ultrasonidos durante 5 minutos. </li><li> Utilizar los imanes para recoger las perlas magnéticas y separar el sobrenadante. </li><li> Añadir 1 ml de SDS al 2%-solución a cada tubo y resuspender utilizando un vórtice. Se hierve durante 5 minutos. </li><li> Recoger las perlas y separar el sobrenadante. Resuspender las perlas en 1 ml de EDTA 0,2 M (pH = 7). </li><li> Incubar los tubos en un baño de ultrasonidos durante 5 minutos y una vez más, separar el sobrenadante. </li><li> Añadir 1 ml de agua a cada tubo y resuspender las perlas. Recoger las perlas y separar el sobrenadante. Repita este paso de lavado dos veces. </li><li> Eliminar el sobrenadante y resuspender las perlas en 1 ml de 10 mM CoCl 2 solución. Colocar en un agitador durante 10 minutos. </li><li> Eliminar el sobrenadante utilizando un imán para recoger las perlas y resuspender en 1 ml de enlace / tampón de lavado. </li><li> Eliminar el sobrenadante y lavar las perlas dos veces utilizando 1 ml de 20% (v / v) de etanol </li><li> Resuspender las perlas de su volumen original de 20% (v / v) ethanol para obtener perlas regeneradas en una concentración de 40 mg / ml. </li></ol> 6. Interpretación de los Resultados <ol><li> Muestra de control 1, en la que se añadió ningún virus radiomarcado, se utiliza para corregir la radioactividad de fondo y para establecer el valor radiactividad 0%. Muestra de control 2, que no contiene nanocuerpos y donde por consiguiente, todos los virus radiomarcado permanece en el sobrenadante, se establece como el valor radiactividad 100%. </li><li> El porcentaje de radiactividad en el sobrenadante (= un%) es una medida de la precipitación total (= 100 - un%) del virus radiomarcado por el nanocuerpos. </li></ol> 7. Los resultados representativos Los resultados representativos para este ensayo de captura de afinidad se muestran en las figuras 2, 3 y 4. En la Figura 2, la afinidad de PVSS38C, una específica para nanocuerpos poliovirus, se muestra. La afinidad de PVSS38C fue probado para tres diferentes antígenos de poliovirus: Nativo-antígeno (N-antígeno), climatizada antígeno (H-antígeno) y las subunidades 14S. N-antígeno es el virus intacto que es infeccioso. Cuando el virus se calienta (20 minutos a 56 ° C) o unida a una superficie sólida (resultados no publicados), la conformación de los cambios cápside, resultando en una pérdida (parcial) del ARN viral y la proteína de la cápside VP4, y vacío cápsides se forman que luego se llama H-antígenos. Subunidades 14S son intermedios de montaje. Estos antígenos son reconocidos por los diferentes conjuntos de anticuerpos después de la vacunación 10 y por lo tanto, poseen epítopos antigénicos y sitios diferentes. En la figura el porcentaje de la radiactividad se encuentra en el sobrenadante se representa como una función de la concentración de Nanobodies. Se puede observar que la radioactividad en el sobrenadante de las muestras que contienen radiomarcados subunidades 14S disminuye con concentraciones crecientes de la PVSS38C nanocuerpos. Esto puede traducirse como un aumento en la cantidad de poliovirus 14S Con subunidadesconectado al PVSS38C nanocuerpos e indirectamente a las perlas magnéticas. El título de la captura (= la concentración de Nanobodies necesario para capturar 50% del virus radiomarcado) puede ser calculada como gráficamente 0.099 nM. N afinidad de PVSS38C para la N-antigénica y H-antigénica forma de poliovirus se observa. A partir de estos datos se interpreta que PVSS38C está interactuando con un epítopo que es exclusivamente presentes en la subunidad 14S y no en las dos formas antigénicas otros de poliovirus. Para demostrar que la pérdida de la radiactividad del sobrenadante es sólo debido a la afinidad específica de la nanocuerpos hacia sus antígenos, el mismo ensayo se realizó con NB1, un nanocuerpos generada contra el regulador transcripcional de lrpB sulfataricus Sulfolobus y se sabe que no tienen ninguna interacción con cualquier antígeno de poliovirus. El resultado de este ensayo se muestra en la Figura 3. Todos los virus radiomarcado permanece en los sobrenadantes que muestran que no hay reactividad deNB1 en contra de las tres formas antigénicas del virus de la polio. Reproducibilidad de los resultados fue probado por repetir el experimento para un total de ocho veces para un determinado Nanobodies (es decir, PVSP29F) en días diferentes y por diferentes personas. Los resultados se muestran en la Figura 4. El valor medio de la radiactividad porcentaje en el sobrenadante se calculó para cada concentración nanocuerpos y está representado en correspondencia con su desviación estándar. <img alt="Figura 1." src="/files/ftp_upload/3937/3937fig1.jpg" /> Figura 1. Una visión general del principio de (A) y el método (B) del ensayo. A. Su-etiquetados nanocuerpos que reconocen específicamente un antígeno determinado poliovirus será capaz de interactuar con las perlas magnéticas recubiertas de cobalto y se precipitará el virus marcado radiactivamente. Nanocuerpos B. Su-etiquetados se añaden a poliovirus marcado radiactivamente y se incubaron. El cobalto-perlas magnéticas recubiertas sonagregado y separación magnética del antígeno unido / no unido se realiza. La radiactividad del sobrenadante y se mide la cantidad de antígeno capturado puede ser derivado. <img alt="Figura 2" src="/files/ftp_upload/3937/3937fig2.jpg" /> Figura 2. Magnética perlas de captura por afinidad de los antígenos de poliovirus diferentes por nanocuerpos PVSS38C. El porcentaje de la radiactividad se encuentra en el sobrenadante se representa como una función de la concentración de PVSS38C. Por 14S una relación concentración-respuesta se puede encontrar, que muestra la interacción de PVSS38C con un epítopo presente exclusivamente en 14S. No hay interacción significativa con N-, ni H-antígeno se puede detectar, aunque un insignificante no específica capturar a concentraciones muy altas se nota. <img alt="Figura 3" src="/files/ftp_upload/3937/3937fig3.jpg" /> Figura 3. Magnética perlas de captura por afinidad de los antígenos de poliovirus diferentes por nanocuerpos NB1 </strong&gt;. El porcentaje de la radiactividad se encuentra en el sobrenadante se representa como una función de la concentración de NB1. NB1 se sabe que tiene ninguna interacción con cualquier virus de la polio-antígeno o subunidad. Desde 100% de la radiactividad se encuentra en el sobrenadante, ningún virus era no unido específicamente a NB1. La pérdida de radiactividad en la figura 2 es por lo tanto, sólo debido a la afinidad específica de la nanocuerpos hacia sus antígenos. <img alt="Figura 4." src="/files/ftp_upload/3937/3937fig4.jpg" /> Figura 4. Reproducibilidad del ensayo. El valor medio de la radiactividad porcentaje en el sobrenadante se representa como una función de la concentración de Nanobodies. El experimento se repitió ocho veces en días diferentes y por diferentes personas. La desviación estándar se representa como barras verticales.

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No tenemos nada que revelar.

En el protocolo, la radiactividad de la interacción con el antígeno nanocuerpos se define como la pérdida de virus radiomarcado a partir del sobrenadante. Por lo tanto la cantidad de radiactividad precipitada (= 100 - un%) (unido a las perlas magnéticas) se puede estimar en una forma indirecta por la radioactividad en el sobrenadante (= un%). Por otro lado, también es posible medir la radiactividad de la fracción precipitada la envolvente de antígeno en una forma particular a eluyendo los inmunocomplejos de las p...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt; Nombre del reactivo</strong</td&gt;<td<strong&gt; Empresa</strong</td&gt;<td<strong&gt; Número de catálogo</strong</td&gt;<td<strong&gt; Comentarios</strong</td&gt;</tr&gt;<tr&gt;<td&gt; Dynabeads Su etiqueta de aislamiento y Jalón</td&gt;<td&gt; Invitrogen</td&gt;<td&gt; 101.03D</td&gt;<td&gt; Las perlas magnéticas</td&gt;</tr&gt;<tr&gt;<td&gt; OptiPhase &#39;HiSafe&#39; 2</td&gt;<td&gt; Perkin Elmer</td&gt;<td&gt; 1200 - 436</td&gt;<td&gt; Centelleo líquido</td&gt;</tr&gt;</tbody&gt;</table

a liquid phase affinity capture assay using magnetic beads to study protein-protein interaction: the poliovirus-nanobody example

En este artículo, un simple, cuantitativo, la afinidad de la fase líquida ensayo de captura se presenta. Siempre que una proteína puede ser marcados y etiquetados otra proteína, este método puede ser aplicado para la investigación de las interacciones proteína-proteína. Se basa en una mano en el reconocimiento de la proteína marcados por perlas magnéticas recubiertas de cobalto y en la otra mano en la interacción entre la proteína marcados y una segunda proteína específica que se etiqueta. En primer lugar, las proteínas marcadas y etiquetada se mezclan y se incuban a temperatura ambiente. Las perlas magnéticas, que reconocen la etiqueta, se añaden y la fracción unida de proteína marcada se separa de la fracción no unida mediante imanes. La cantidad de proteína marcada que es capturada se puede determinar de una manera indirecta mediante la medición de la señal de la proteína marcada se mantuvo en la fracción no unida. La fase líquida se describe ensayo de afinidad es muy útil cuando las proteínas conformacionales de conversión son sensibles culoAyed. El desarrollo y la aplicación del ensayo se demuestra por la interacción entre poliovirus y poliovirus reconociendo nanocuerpos 1. Dado que el poliovirus es sensible a la conversión conformacional 2 cuando está unido a una superficie sólida (resultados no publicados), el uso de ELISA es limitado y un sistema basado en fase líquida, por tanto, se prefiere. Un ejemplo de un sistema de fase líquida basada utiliza a menudo en polioresearch 3,4 es la proteína de micro-inmunoprecipitación Un ensayo 5. A pesar de que esta prueba ha demostrado su aplicabilidad, se requiere un Fc-estructura, que está ausente en los Nanobodies 6,7. Sin embargo, como otra oportunidad, estos interesantes y estable de un solo dominio anticuerpos 8 puede ser fácilmente diseñado con diferentes etiquetas. El ampliamente utilizado (Su) 6-etiqueta muestra afinidad por los iones bivalentes tales como níquel o cobalto, que puede a su vez, ser fácilmente recubierta por perlas magnéticas. Por lo tanto, desarrolló este cuantitativo simpleensayo de afinidad de captura sobre la base de cobalto perlas magnéticas recubiertas. Poliovirus se marcó con 35 S para permitir una interacción sin obstáculos con los Nanobodies y para hacer una detección cuantitativa factible. El método es fácil de realizar y puede establecerse con un bajo costo, que se ve apoyada por la posibilidad de regenerar eficazmente las perlas magnéticas.

Watch this Scientific Journal Video about A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example at JoVE.com

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

En este artículo, un simple, cuantitativo, la afinidad de la fase líquida ensayo de captura se presenta. Es una técnica fiable basado en la interacción entre partículas magnéticas y las proteínas marcadas (por ejemplo nanocuerpos) en una mano y la afinidad entre la proteína marcados y una segunda proteína, con la etiqueta (por ejemplo, virus de la polio) en el otro.

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 ...

a-liquid-phase-affinity-capture-assay-using-magnetic-beads-to-study

Research

JoVE Journal

Biology

11.8K vistas.  Vrije Universiteit Brussel. En este artículo, un simple, cuantitativo, la afinidad de la fase líquida ensayo de captura se presenta. Es una técnica fiable basado en la interacción entre partículas magnéticas y las proteínas marcadas (por ejemplo nanocuerpos) en una mano y la afinidad entre la proteína marcados y una segunda proteína, con la etiqueta (por ejemplo, virus de la polio) en el otro.

Video: A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Utilizando el sistema de fraccionamiento GELFREE 8100 de peso molecular basado en Fraccionamiento con la recuperación de la fase líquida

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

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 ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

Lípidos vesículas mediada por cromatografía de afinidad utilizando magnética clasificación de células activadas (LIMACS): un nuevo método para analizar la interacción de proteínas y lípidos

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.

An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.

La conversión de un ELISA de captura para un ensayo de Luminex xMAP utilizando un anticuerpo Multiplex Método de detección

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

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

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

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

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

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

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

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

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

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 ...

Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron regulatory proteins, IRP1 and IRP2. They bind to iron responsive elements (IREs) within the untranslated regions (UTRs) of several target mRNAs, thereby controlling the mRNAs translation or stability. IRE/IRP interactions have been widely studied by EMSA. Here, we describe the EMSA protocol for analyzing the IRE-binding activity of IRP1 and IRP2, which can be generalized to assess the activity of other RNA-binding proteins as well. A crude protein lysate containing an RNA-binding protein, or a purified preparation of this protein, is incubated with an excess of32 P-labeled RNA probe, allowing for complex formation. Heparin is added to preclude non-specific protein to probe binding. Subsequently, the mixture is analyzed by non-denaturing electrophoresis on a polyacrylamide gel. The free probe migrates fast, while the RNA/protein complex exhibits retarded mobility; hence, the procedure is also called &#8220;gel retardation&#8221; or &#8220;bandshift&#8221; assay. After completion of the electrophoresis, the gel is dried and RNA/protein complexes, as well as free probe, are detected by autoradiography. The overall goal of the protocol is to detect and quantify IRE/IRP and other RNA/protein interactions. Moreover, EMSA can also be used to determine specificity, binding affinity, and stoichiometry of the RNA/protein interaction under investigation.

Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

Here we present a protocol to analyze RNA/protein interactions. The electrophoretic mobility shift assay (EMSA) is based on the differential migration of RNA/protein complexes and free RNA during native gel electrophoresis. By using a radiolabeled RNA probe, RNA/protein complexes can be visualized by autoradiography.

La movilidad electroforética cambio de ensayo (EMSA) para el estudio de las interacciones RNA-proteína: El IRE / IRP Ejemplo

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron ...

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 ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.