Estamos muy agradecidos a Frank Ervin, Palmour Roberta y el personal de Laboratorios de Ciencias de la Conducta fundación con sede en San Cristóbal, de las Indias Occidentales, por su continuo apoyo a nuestro trabajo de primates. Este trabajo fue apoyado por una beca de la Fundación X Frágil de Investigación de Canadá (FXRFC) a SZ y - subvenciones de funcionamiento de los Institutos Canadienses de Investigación en Salud (AC) y la Nacional de Ingeniería y de Investigación de Canadá (MP).

Inmunohistoquímica es una de las técnicas más utilizadas para la caracterización de la expresión de proteínas en el cerebro de los diversos modelos de experimentación animal. Es relativamente fácil de llevar a cabo procedimientos sistemáticos de inmunohistoquímica en el cerebro de los roedores y otros modelos experimentales común con el tamaño del cerebro similar. Sin embargo, no hay ningún trabajo publicado en nuestro conocimiento que proporciona una explicación detallada de cómo llevar a cabo dichos procedimientos de inmunodetección a través de un cerebro de mono entero. Lo que sigue es una descripción detallada de cómo preparar un cerebro del mono todo a gran escala la detección inmunohistoquímica de las proteínas diana. Este trabajo ha surgido como resultado de la colaboración entre las actividades comerciales y académicas. Por lo tanto, los detalles relativos a los tejidos inclusión y corte sigue siendo un conocimiento de su propiedad de la NSA. Parte 1: El tratamiento de los animales y la preparación del tejido El cerebro de un adulto mono verde (Cercopithecus aethiops) se utiliza para el presente Protocolo. Todos los procedimientos se llevan a cabo de acuerdo con el Consejo Canadiense de Protección de los Animales (CCPA) Directrices para el uso y cuidado de los animales en la investigación biomédica 1. <ol><li> Animal está profundamente sedado con clorhidrato de ketamina (10 mg / kg, im), sacrificados con una sobredosis de pentobarbital sódico (25 mg / kg, iv) y perfundidos transcardially con PBS 0,1 M hasta que esté completamente desangrado. </li><li> Esto es seguido por una solución de paraformaldehído al 4% en PBS durante 5 minutos (aproximadamente 1 litro). </li><li> El cerebro se coloca en externalizados calificado soluciones de sacarosa PBS buffer (10, 20 y 30%, en la secuencia), que contiene 0,02% de ácido sódico y se mantiene a 4 ˚ C hasta que el cerebro se hunde hasta el fondo del recipiente. La solución es reemplazado cada pocos días hasta que el cerebro sufre congelación y corte. </li><li> El cerebro se envía a los Asociados de la neurociencia (NSA, Knoxville, TN) para ser sometido a su propia MultiBrain ™ inclusión y de congelación y corte la tecnología. </li><li> Siete (7) puntos de referencia de alineación se colocan en la matriz de incrustación para que las secciones se pueden orientar adecuadamente (es decir, de izquierda a derecha orientación) y re-alineados tras el procesamiento histológico se ha completado. </li><li> El bloque congelado es fotografiados digitalmente en la cara del bloque antes de cada sección de serie se obtiene de la manzana. Las imágenes digitales, así como las secciones de este cerebro puede ser utilizada después para la reconstrucción 3D de los mapas anatómicos e histológicos, respectivamente. </li><li> Serie de libre flotación secciones coronal, con un espesor de 50 micras por cada sección, se obtienen de todo el cerebro. </li></ol> Parte 2: procesamiento histológico Las secciones son elegidos en un determinado intervalo espacial (por ejemplo, de 500 micras) y para nuestros propósitos experimentales se procesaron con los siguientes anticuerpos: X frágil retraso mental proteína (FMRP, Chemicon, Temecula, CA), SMI32 (Sternberger monoclonales Inc.; Baltimore, MD), y NeuN (Chemicon, Temecula, CA). FMRP es una proteína citoplasmática que encuentra en abundancia en las neuronas de los cerebros de transporte normal y permutación 2. NeuN (núcleos neuronales) reconoce específicamente la unión al ADN específica de neuronas NeuN proteína que está presente en la mayoría de las neuronas y se distribuye en núcleos neuronales, perikarya y algunos procesos neuronales proximal 3. SMI-32 es un anticuerpo monoclonal que reconoce el epítopo no fosforilada, el 4 de proteínas de neurofilamentos. <ol><li> En un intervalo de 500 m, aproximadamente 140 secciones son utilizadas por anticuerpos. </li><li> Todas las incubaciones, se lava las manchas y los pasos se llevan a cabo con agitación suave con platos especializados manchas y cestas (HistoTools, Knoxville, TN). </li><li> Las secciones se incuban primero en una solución que contiene 0,1 M PBS/0.3% Triton X-100 (TX) / 5% de suero normal ronca (NHS) durante 60 minutos con el fin de reducir la unión no específica de moléculas de anticuerpos y la tinción de fondo resultante. </li><li> Las secciones marcadas para inmunoticción FMRP someterse a una operación adicional de recuperación de antígenos mediante el uso de solución de antígeno Desenmascarando (Vector Labs, Burlingame, CA) de acuerdo a las instrucciones del proveedor. </li><li> Las secciones se incuban durante la noche y en días distintos en sus soluciones de anticuerpos respectivos que contiene 0,1 M PBS/0.3% TX / NHS 3% (con un factor de dilución de 1 / 5, 000 para FMRP y SMI-32 y 1 / 2, 000 para los anticuerpos NeuN. </li><li> Al día siguiente, las secciones se lavan durante tres períodos de 10 minutos en la solución de lavado (0.1 M PBS/0.3% TX), seguido de una incubación de 2 horas a temperatura ambiente en biotinilado anti-anticuerpo secundario del ratón se crió en caballo (Vector Labs, 1 / 1000 dilución en 0,1 M PBS/0.3% NHS TX / 3%). </li><li> Después de una nueva serie de 3 a 10 min lavados, las secciones se colocan en una solución de complejo avidina-biotina conjugada con peroxidasa (Vector Labs) durante 1 hora a temperatura ambiente. </li><li> Finalmente, después de otra serie de lavados, las secciones son sometidas durante 10 minutos a un diámetro mayor de níquelminobenzidine (Ni-DAB) la reacción que produce una mancha de color azul oscuro dentro de las neuronas inmunorreactivas. </li><li> Al finalizar el procedimiento histológico, las secciones se montan en placas de vidrio recubiertas de gelatina. </li><li> Todas las secciones se secan al aire durante la noche y coverslipped acuerdo con los procedimientos estándar. </li></ol> Parte 3: Resultados del representante: Este método produce un perfil de expresión completa de una proteína diana de interés a través de un cerebro de mono entero. Aquí se muestra representativa secciones coronal que proporcionan una instantánea de la FMRP, NeuN y SMI32 expresión en el cerebro del mono mismo. <img src="/files/ftp_upload/1286/1286fig1.png" alt="" /> Figura 1: Plato de tinción (A) y la cesta (B) utilizados para el procesamiento por lotes a gran escala de libre flotación secciones para la inmunodetección. <img src="/files/ftp_upload/1286/1286fig2.png" alt="" /> Figura 2: Representante de las secciones coronal de un cerebro monos verdes teñidas de FMRP (A), NeuN (B) y los anticuerpos SMI32 (C).

<ol>
	<li>Olfert, E. D., Cross, B. M., McWilliam, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide to the Care and Use of Experimental Animals. , (1993).</li><li>Devys, D., Lutz, Y., Rouyer, N., Bellocq, J. P., Mandel, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+FMR-1+protein+is+cytoplasmic,+most+abundant+in+neurons+and+appears+normal+in+carriers+of+a+fragile+X+premutation.">The FMR-1 protein is cytoplasmic, most abundant in neurons and appears normal in carriers of a fragile X premutation.</a> Nat Genet. 4, 335-340 (1993).</li><li>Mullen, R. J., Buck, C. R., Smith, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NeuN,+a+neuronal+specific+nuclear+protein+in+vertebrates.">NeuN, a neuronal specific nuclear protein in vertebrates.</a> Development. 116, 201-211 (1992).</li><li>Sternberger, L. A., Sternberger, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+distinguish+phosphorylated+and+nonphosphorylated+forms+of+neurofilaments+in+situ.">Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ.</a> Proc Natl Acad Sci U S A. 80, 6126-6130 (1983).</li></ol>

The authors have nothing to disclose.

Hay dos pasos fundamentales en este procedimiento que hacen a gran escala de detección de proteínas en el cerebro de un mono completo posible. Uno de ellos es el protocolo de inclusión y corte, que sigue siendo el conocimiento de su propiedad de la NSA. El otro es el uso de platos de tinción y cestas proporcionada por HistoTools. Este último permite un manejo fácil y rápido de muchas (~ 40) secciones en un momento dado. También proporciona los medios para tratar de manera uniforme a todos los sectores a través ...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>anti-FMRP monoclonal antibody</td>
<td>&nbsp;</td>
<td>Chemicon</td>
<td>MAB2160</td>
<td>Requires antigen retrieval in fixed tissue.</td>
</tr>
<tr>
<td>anti-NeuN monoclonal antibody</td>
<td>&nbsp;</td>
<td>Chemicon</td>
<td>MAB377</td>
<td>N/A</td>
</tr>
<tr>
<td>anti-Neurofilament H monoclonal antibody</td>
<td>&nbsp;</td>
<td>Sternberger Monoclonals Inc.</td>
<td>SMI32</td>
<td>N/A</td>
</tr>
<tr>
<td>Antigen Unmasking Solution</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>H-3300</td>
<td>N/A</td>
</tr>
<tr>
<td>Normal horse serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16050122</td>
<td>500 mL</td>
</tr>
<tr>
<td>Biotinylated Anti-Mouse IgG (H+L), made in horse</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>BA-2000</td>
<td>&nbsp;</td>
<tr>
<td>VECTASTAIN ABC Kit (Standard)</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>PK-4000</td>
<td>1.5 mg</td>
</tr>
<tr>
<td>Staining dish for floating sections</td>
<td>&nbsp;</td>
<td>HistoTools</td>
<td>10009</td>
<td>12 x 65 mm</td>
</tr>
<tr>
<td>Staining transport basket</td>
<td>&nbsp;</td>
<td>HistoTools</td>
<td>10012</td>
<td>large 
(fits 12 x 65 mm dish)</td>
</tr>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>P8514B</td>
<td>N/A</td>
</tr>
<tr>
<td>DAB</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>AC11209-0050</td>
<td>N/A</td>
</tr>		</tbody>
		</table>
		</div>

batch immunostaining for large-scale protein detection in the whole monkey brain

Inmunohistoquímica (IHC) es una de las técnicas de laboratorio más utilizados para la detección de proteínas diana<em&gt; In situ</em&gt;. Las cuestiones relativas a los patrones de expresión de una proteína diana a través de todo el cerebro es relativamente fácil de responder cuando se utiliza IHC en los cerebros pequeños, como los de los roedores. Sin embargo, responder las mismas preguntas en un cerebro grande y complicado, como las de los primates presentan una serie de desafíos. Aquí se presenta un enfoque sistemático para la inmunodetección de las proteínas diana en el cerebro de un mono adulto. Este enfoque se basa en el tejido de inclusión y corte metodología de Asociados Neurociencias (NSA), así como herramientas desarrolladas específicamente para la tinción de lotes de libre flotación secciones. Es el resultado de la tinción uniforme de un conjunto de secciones que, en un intervalo particular, representa a todo el cerebro. Las secciones resultantes manchado puede ser sometido a una amplia variedad de procedimientos analíticos para medir los niveles de proteína, la población de neuronas que expresan una determinada proteína.

Watch this Scientific Journal Video about Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain at JoVE.com

Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

A gran escala inmunodetección de las proteínas diana a través del cerebro de los primates todo es posible mediante el empleo de tejidos novedosos métodos de inclusión y corte en combinación con el uso de aparatos creativas para la tinción de lotes de varias secciones de flotación libre en un momento dado.

Lote de inmunotinción para la detección de proteínas a gran escala en el cerebro del mono entero

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the ...

batch-immunostaining-for-large-scale-protein-detection-whole-monkey

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

12.6K vistas.  Montreal Neurological Institute. A gran escala inmunodetección de las proteínas diana a través del cerebro de los primates todo es posible mediante el empleo de tejidos novedosos métodos de inclusión y corte en combinación con el uso de aparatos creativas para la tinción de lotes de varias secciones de flotación libre en un momento dado.

Video: Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

A gran escala de pantallas de bibliotecas metagenómicas

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

Investigación

Biología

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

La detección de ubiquitinación de proteínas

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

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 ...

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

La purificación de la M. magneticum Tensión AMB-1 magnetosomas proteína asociada MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to mammals, zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

A gran escala basado en pez cebra En vivo Pantalla de moléculas pequeñas

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to ...

Infinium Assay for Large-scale SNP Genotyping Applications

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina&#39;s panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.

A protocol is described that uses Illumina&#39;s Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.

Ensayo Infinium para aplicaciones a gran escala de genotipado de SNP

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

Una estrategia para sensibles, grande escala cuantitativa Metabolómica

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

La investigación de las interacciones proteína-proteína en las células vivas utilizando la transferencia de energía de resonancia de bioluminiscencia

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, log-phase, and stationary-phase. Each growth phase can reveal different aspects of fitness that are related to various environmental and genetic conditions. High-resolution and quantitative measurements of all 3 phases of growth are generally difficult to obtain. Here we present a detailed method to characterize all 3 growth phases on solid media using an assay called One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). ODELAY quantifies growth phenotypes of individual cells growing into colonies on solid media using time-lapse microscopy. This method can directly observe population heterogeneity with each growth parameter in genetically identical cells growing into colonies. This population heterogeneity offers a unique perspective for understanding genetic and epigenetic regulation, and responses to genetic and environmental perturbations. While the ODELAY method is demonstrated using yeast, it can be utilized on any colony forming microorganism that is visible by bright field microscopy.

We present a method for quantifying growth phenotypes of individual yeast cells as they grow into colonies on solid media using time-lapse microscopy termed, One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). Population heterogeneity of genetically identical cells growing into colonies can be directly observed and quantified.

ODELAY: Un método a gran escala para la cuantificación multiparamétrica del crecimiento de la levadura

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

Identificación de nuevos reguladores de la transpiración de plantas mediante el cribado térmico de imágenes a gran escala en Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...