Este trabajo fue apoyado por becas de la Fundación Canadiense para la Innovación (CFI), el Instituto Canadiense de Investigación en Salud (CIHR), y el de Ciencias Naturales e Ingeniería de Investigación de Canadá (NSERC).

Parte 1: La interrupción de la Influenza A virión <ol><li> Añadir 750 l de tampón SM (20 mM MES, 150 mM NaCl, 30 mM Tris, pH 7,5) en un tubo de centrífuga Beckman policarbonato (11 mm x 34 mm) diseñado para encajar en un rotor TLA-120.2 para su uso en un Beckman Optima Max-E ultracentrífuga. </li><li> Añadir 500 l de la gripe A (H3N2 X-31 A/AICHI/68 cepa, 2 mg / ml) en ese tubo. Mezcla del virus con el buffer de MNT pipeteando arriba y abajo varias veces. </li><li> Centrifugar durante 10 minutos a 109.000 xg, 4 º C, en un Beckman Optima Max-E centrífuga con un rotor TLA-120.2. </li><li> Eliminar el sobrenadante y resuspender el precipitado en 500 buffer interrupción l (100 mM KCl, 5 mM MgCl 2, 5% (w / v) de glicerol, 50 mM octilglucósido, 10 mg / ml lisolecitina, 1,5 mM de ditiotreitol 100 mM MES, pH 5,5). </li><li> Agitar vigorosamente, y se agita a 31 ° C durante 20 minutos en un termomezclador eppendorf. </li></ol> Parte 2: gradiente de glicerol sedimento centrifugado de velocidad <ol><li> Preparar un gradiente de glicerol mediante la colocación de las siguientes cantidades de glicerol en un tubo de centrífuga Beckman ultraclaros (13 mm x 51 mm): 1 ml al 70% (v / v) de glicerol, 0,75 ml de glicerol al 50%, 0.375 ml de glicerol 40%, y 1,8 ml 33% de glicerol. Soluciones de glicerol se obtienen mezclando glicerina pura con tampón NM (150 mM NaCl, 50 mM MES, pH 5,5). También preparan un tubo que contiene tanto el equilibrio de glicerol y tampón. </li><li> Vortex la muestra viral interrumpe de nuevo, y cargarlo en el gradiente de glicerol. </li><li> Centrifugar el gradiente de 3,75 horas a 217.000 xg, 4 º C, en un Beckman MLS-50-Rotor oscilante. </li><li> Después de la centrifugación es completa, de forma manual recoger 250 alícuotas de la pendiente a partir de la parte superior del tubo. Mantenga las fracciones en hielo oa 4 º C. </li></ol> Parte 3: Análisis de las fracciones de gradiente de glicerol por electroforesis en gel de poliacrilamida SDS <ol><li> Retire 20 l de cada fracción a un tubo nuevo. </li><li> Añadir 5 l de tampón 5x muestra de SDS-PAGE. </li><li> Calor a 95 ° C durante 5 minutos. </li><li> Centrifugar las muestras de forma breve y cargarlos en un gel de poliacrilamida al 10%, e incluyen marcadores de peso molecular en uno de los pozos. </li><li> Ejecutar las muestras en el gel hasta que el colorante azul de bromofenol carga llega a cerca de la parte inferior del gel. </li><li> Tinción del gel con azul de Coomassie. </li></ol> Parte 4: concentración de las fracciones vRNP <ol><li> Elija las fracciones de glicerol que contiene principalmente NP, combinarlos, y los distribuyen en dos tubos de centrífuga Beckman policarbonato (11 mm x 34 mm). </li><li> Llene cada tubo con dietil pirocarbonato (DEPC) tratados con agua ultrapura, y una pipeta de arriba a abajo varias veces para mezclar. </li><li> Centrifugar durante 4,5 horas a 157.000 xg, 4 º C, en un Beckman TLA-120.2 del rotor. </li><li> Eliminar el sobrenadante y resuspender el precipitado en 50 l de agua tratada con DEPC. </li><li> Si lo desea, el 280 A de la vRNPs concentrado se puede medir. Como NP es el principal componente presente en las fracciones reunidas, una estimación de la molaridad de NP se puede calcular utilizando el coeficiente de extinción de 55350 M -1 cm -1 (según lo determinado por ProtParam 3). </li><li> Alícuota del vRNPs purificada, y almacenarlas congeladas a -80 ° C para su uso posterior. </li></ol> Parte 5: tinción negativa de vRNPs <ol><li> Diluir 1 l de agua purificada vRNP en 9 l de recién hecho y se filtra el doble buffer MNT (20 mM MES, 150 mM NaCl, 30 mM Tris, pH 7,5). Otros tampones también están bien para diluir el vRNPs y para lavar la red de la muestra (paso 5.5), pero no el uso de acetato de uranilo PBS si se utiliza para la tinción negativa. </li><li> Glow-descarga una muestra (TEM cobre) la red previamente recubierto de una película de carbono Parlodión y durante 30 segundos. </li><li> Sostenga el recién resplandor descargada la red muestra con pinzas, y aplicar una gota de 5 l de vRNP diluido en la cuadrícula de la muestra. </li><li> Salir de la gota de muestra en la parrilla durante 8 minutos. </li><li> Mientras espera, coloque una pequeña tira de Parafilm en el banco, y prescindir de dos gotas que contienen 10 l cada uno de tampón SM (para lavar la red), y una caída de 80 l de la solución de tinción recién hecho (1% molibdato de amonio o una % de acetato de uranilo) en el Parafilm. </li><li> Después de 8 minutos de adsorción, la mecha a una parte de la solución de la muestra de la red muestra con un pedazo de papel de filtro (cortado en triángulos). No permita que la red muestra que se seque. </li><li> Lave la red muestra en dos gotas que contienen 10 l cada uno de búfer SM por un tiempo total de 1 minuto. Esto se hace con cuidado bajar de la red de la muestra en la caída, y luego absorción de parte de la solución de la muestra de la red muestra con un pedazo de papel de filtro (sin dejar que la red de muestra seca). </li><li> Inmediatamente después de la absorción de la última gota de buffer de la red muestra, completasumergir mente la muestra dentro de la red de la gota mancha y espere un minuto. </li><li> Completamente la mecha de la solución de tinción de la red muestra con un pedazo de papel de filtro. </li><li> Permitir la red muestra se seque al aire durante varios minutos antes de la observación en el microscopio electrónico de transmisión. </li></ol> Parte 6: Resultados del representante: <img src="/files/ftp_upload/old/1105_2009_1_22_1/1105_Figure2.png" alt="Figura 2" width="600" height="215" /> Figura 1 Como la gripe A NP (~ 56 kDa) es la principal proteína en los complejos de ribonucleoproteína viral, la banda de NP en general es el más fuerte de banda en las fracciones que contiene el vRNPs (Figura 1). Además de NP, cada vRNP influenza también contiene una copia de un trimérica RNA polimerasa (pesos moleculares de kDa 82, 86 y 86.5) complejos. Estos pueden ser o no ser visible por tinción con Coomassie azul debido a su abundancia es baja comparada con la de NP. Las polimerasas, sin embargo, se puede detectar si en lugar de un azul Commassie mancha, una mancha de plata del gel se realiza. La influenza proteína de la matriz M1 (~ 28 kDa) debe ser mínimamente presente en las fracciones en las fracciones de proteína NP pico están presentes. <img src="/files/ftp_upload/old/1105_2009_1_22_2/1105_3.png" alt="Figura 3" width="600" height="360" /> Figura 2 VRNPs negativamente manchado tal como puede verse en un microscopio electrónico de transmisión debe ceder vRNPs que se asemejan a las partículas en forma de barra con longitud variable que son aproximadamente 30 nm a 120 nm de longitud (Figura 2a). El NP oligoméricas está organizado como una cadena de moléculas de NP que se ve doblada en una estructura de doble hélice repetir, por lo que los bucles en ambos extremos de estas partículas en forma de barra a veces se puede ver (figura 2b).

<ol>
	<li>Kemler, I., Whittaker, G., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+import+of+microinjected+influenza+virus+ribonucleoproteins.">Nuclear import of microinjected influenza virus ribonucleoproteins.</a> Virology. 202, 1028-1033 (1994).</li><li>Wu, W. W. H., Weaver, L. L., Pant&#233;, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+analysis+of+the+nuclear+localization+sequences+on+influenza+a+ribonucleoprotein+complexes.">Ultrastructural analysis of the nuclear localization sequences on influenza a ribonucleoprotein complexes.</a> J Mol Biol. 374, 910-916 (2007).</li><li>Gasteiger, E., Walker, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Proteomics Protocols Handbook. , 571-607 (2005).</li><li>Wu, W. W. H., Sun, Y. H. B., Pant&#233;, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+import+of+influenza+A+viral+ribonucleoprotein+complexes+is+mediated+by+two+nuclear+localization+sequences+on+viral+nucleoprotein.">Nuclear import of influenza A viral ribonucleoprotein complexes is mediated by two nuclear localization sequences on viral nucleoprotein.</a> Virol J. 4, 49-49 (2007).</li><li>Babcock, H. P., Chen, C., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+single-particle+tracking+to+study+nuclear+trafficking+of+viral+genes.">Using single-particle tracking to study nuclear trafficking of viral genes.</a> Biophys J. 87, 2749-2758 (2004).</li></ol>

La purificación de vRNPs se basa en el procedimiento descrito por Kemler et al. (1994). 1 Nosotros y otros han utilizado también este protocolo para aislar vRNPs para estudiar su importación nuclear. 2,4,5 Se recomienda el uso de RNasa libre de puntas y los tubos de la hora de manipular vRNPs debido a que el genoma viral se compone de ARN, y por lo tanto, se degrada fácilmente en la presencia de ARN. Además, todos los buffers deben hacerse en el agua que e...

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		<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>H3N2 X-31 A/AICHI/68 influenza A</td>
<td>&nbsp;</td>
<td>Charles River Laboratories</td>
<td>490715</td>
<td>&nbsp;</td>
<tr>
<td>Tris</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T1503</td>
<td>&nbsp;</td>
<tr>
<td>MES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M-8250</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>G33-1</td>
<td>&nbsp;</td>
<tr>
<td>Octylglucoside</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>O-8001</td>
<td>&nbsp;</td>
<tr>
<td>Lysolecithin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L-4129</td>
<td>&nbsp;</td>
<tr>
<td>Dithiothreitol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D-9779</td>
<td>&nbsp;</td>
<tr>
<td>Coomassie brilliant blue G-250</td>
<td>&nbsp;</td>
<td>Kodak</td>
<td>1367796</td>
<td>&nbsp;</td>
<tr>
<td>diethyl pyrocarbonate (DEPC)-treated water</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>750024</td>
<td>&nbsp;</td>
<tr>
<td>Uranyl Acetate</td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>19481</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium Molybdate</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>A-674</td>
<td>&nbsp;</td>
<tr>
<td>Optima MAX-E Ultracentrifuge</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>434491</td>
<td>&nbsp;</td>
<tr>
<td>MLS-50 Rotor Package, Swinging Bucket</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>367280</td>
<td>&nbsp;</td>
<tr>
<td>TLA-120.2 Rotor Assembly, Fixed-Angle, Titanium</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>362046</td>
<td>&nbsp;</td>
<tr>
<td>Eppendorf Thermomixer</td>
<td>&nbsp;</td>
<td>Brinkman</td>
<td>022670000</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

purification and visualization of influenza a viral ribonucleoprotein complexes

La gripe A genoma viral se compone de ocho en sentido negativo, de cadena sencilla moléculas de ARN, envasadas individualmente con varias copias de la influenza A nucleoproteína (NP) en partículas virales ribonulceoprotein (vRNPs). El vRNPs influenza están encerrados dentro de la envoltura viral. Durante la entrada de la celda, sin embargo, estos complejos vRNP son liberados en el citoplasma, donde el acceso a los mecanismos de acogida de transporte nuclear. Para el estudio de la importación nuclear de vRNPs influenza y la replicación del genoma de la gripe, es útil para trabajar con vRNPs aisladas para que los otros componentes del virus no interferir con estos procesos. Aquí se describe un procedimiento para purificar estas vRNPs del virus de la influenza. El procedimiento se inicia con la ruptura de la influenza A virión con detergentes a fin de liberar los complejos vRNP del virión con envoltura. El vRNPs se separan de los otros componentes de la influenza A virión en un gradiente de glicerol 33-70% de velocidad de sedimentación discontinua. Las fracciones obtenidas del gradiente de glicerol se analizan a través de la SDS-PAGE después de la tinción con azul de Coomassie. Las fracciones que contienen NP pico después se agruparon y se concentraron por centrifugación. Después de la concentración, la integridad de la vRNPs se verifica mediante la visualización de la vRNPs por microscopía electrónica de transmisión después de la tinción negativa. La purificación del gradiente de glicerol es una modificación de que a partir de Kemler<em&gt; Et al.</em&gt; (1994)<sup&gt; 1</sup&gt;, Y la tinción negativa ha sido realizada por Wu<em&gt; Et al.</em&gt; (2007).<sup&gt; 2</sup

Watch this Scientific Journal Video about Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes at JoVE.com

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

El genoma del virus de influenza A se compone de ocho complejos separados de ARN y proteínas, denominadas complejos de ribonucleoproteína viral (vRNPs). Este trabajo describe la purificación gradiente de glicerol y de microscopía electrónica de transmisión de visualización de la gripe A vRNPs.

Purificación y visualización de la gripe A ribonucleoprotein complejos virales

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A ...

purification-visualization-influenza-viral-ribonucleoprotein

Research

JoVE Journal

Biology

12.9K vistas.  University of British Columbia - UBC. El genoma del virus de influenza A se compone de ocho complejos separados de ARN y proteínas, denominadas complejos de ribonucleoproteína viral (vRNPs). Este trabajo describe la purificación gradiente de glicerol y de microscopía electrónica de transmisión de visualización de la gripe A vRNPs.

Video: Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

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

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

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

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

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

Investigación

Biología

Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for magnetic resonance imaging (MRI). Many commercially available ligands are especially useful because they contain 
functional groups that allow for fast, high-purity, and high-yielding conjugation to macromolecules and biomolecules via amine-reactive 
activated esters and isothiocyanate groups or thiol-reactive maleimides. While metalation of these ligands is considered common 
knowledge in the field of bioconjugation chemistry, subtle differences in metalation procedures must be taken into account when 
selecting metal starting materials. Furthermore, multiple options for purification and characterization exist, and selection of the most 
effective procedure partially depends on the selection of starting materials. These subtle differences are often neglected in published 
protocols. Here, our goal is to demonstrate common methods for metalation, purification, and characterization of lanthanide complexes 
that can be used as contrast agents for MRI (Figure 1). We expect that this publication will enable biomedical scientists to incorporate 
lanthanide complexation reactions into their repertoire of commonly used reactions by easing the selection of starting materials and 
purification methods.

We demonstrate the metalation, purification, and characterization of lanthanide complexes. The complexes described here can be conjugated to macromolecules to enable tracking of these molecules using magnetic resonance imaging.

Preparación, purificación y caracterización de los complejos lantánidos para el uso de agentes de contraste para resonancia magnética

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

La purificación de Hsp104, una proteína Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

Purificación por cromatografía de ribosomas de levadura de gran actividad

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

Método para el aislamiento e identificación de mRNAs, microRNAs y componentes proteicos de ribonucleoprotein complejos de extractos celulares usando RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

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

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

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

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

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

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

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

Cartográficas basadas en AFM de las propiedades elásticas de las paredes celulares: en tejido, celular, y las Resoluciones Subcelulares

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

Aislamiento rápido y purificación de mitocondrias para el trasplante por disociación de tejidos y filtración diferencial

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

Generación y purificación de Humanos INO80 remodelación de la cromatina complejos y Subcomplejos

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

Validación de un modelo de ratón para interrumpir LINC Complejos en una celda de manera específica

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...