Agradecemos el apoyo técnico de Sini Miettinen, Minna Pöllänen y Taru Rautavesi. Agradecemos Helka Nurkkala para proporcionar células HEK 293 que expresan de etiquetado doble Strep GFP y Pekka Evijärvi para el suministro de equipos de grabación de sonido. Este trabajo fue financiado por la Academia de Finlandia, conceder los números 138329, 134684 y 258978.

1. La reticulación de Strep-Tactin polimetacrilato de resina con Bis (sulfosuccinimidil) suberato (BS3) <ol><li> Equilibrar un microtubo sellado que contiene 2 mg de BS3 agente de reticulación a temperatura ambiente. PRECAUCIÓN: BS3 es una sustancia peligrosa. Utilice guantes y gafas protectoras. </li><li> Resina de polimetacrilato Resuspender Strep-Tactin (suspensión al 50% en 100 mM de Tris-HCl, pH 8,0; EDTA 1 mM; NaCl 150 mM) mediante una breve agitación vigorosa y transferir inmediatamente 600 l de la suspensión a una columna de centrifugación usando una punta de pipeta con la extremo cortado. </li><li> Centrifugar a 1500 xg durante 30 segundos a temperatura ambiente. Desechar el flujo a través y añadir 450 l de tampón fosfato salino (PBS) a la columna. </li><li> Repita el paso anterior 2 veces más para reemplazar completamente el tampón Tris con PBS y dejar la resina en 430 l de PBS, ajustada a pH 8,0 después de la última centrifugación. </li><li> Perfore el papel aluminio de la microtubo con BS3 con una punta de pipeta que contiene 1001; l de agua ultrapura. Disuelva el polvo en agua BS3 pipeteando suavemente hacia arriba y hacia abajo y añadir inmediatamente 20 l de la solución a la columna de centrifugación. La concentración final de BS3 en la reacción de reticulación es de ~ 1,2 mM. </li><li> Girar la columna durante 30 min a temperatura ambiente. Compruebe que la resina se mezcla adecuadamente con la solución de BS3. </li><li> Para interrumpir la reacción, añadir 6 l de 3M Tris-HCl, pH 7,5 y girar la columna durante otros 15 min a temperatura ambiente. </li><li> Centrifugar a 1500 xg durante 30 segundos a temperatura ambiente. Desechar el flujo a través y resuspender la resina reticulada en 450 l de solución salina tamponada con Tris con Tween 20 (TBST). Repetir la centrifugación y etapas de lavado dos veces más. En el último paso, resuspender la resina en 450 l de TBS. </li><li> Transferir la suspensión de resina a un tubo nuevo usando una punta de pipeta con el extremo cortado. Resuspender la resina que queda en la columna con otros 450 l de TBS y transferir al mismo tubo. Repita ªe último paso una vez más para asegurar la transferencia máxima de la resina de la columna al tubo. </li><li> Deje que el tubo de reposar durante 10 minutos a temperatura ambiente y ajustar el volumen a 600 l, eliminando el exceso de TBS. La resina está lista para su uso inmediato (recomendado) o se puede almacenar a 4 ° C, sin congelar. </li></ol> 2. Unión de la proteína cebo etiquetados-Twin-Strep y Associated Complejos para el reticulado Strep-Tactin Polimetacrilato Resina <ol><li> Centrifugar 1 ml de la muestra de proteína cebo en tampón de unión a 17000 xg durante 10 min a 4 ° C y transferir el sobrenadante a un tubo nuevo. </li><li> Para minimizar la unión de proteínas biotiniladas endógenos a la resina de Strep-Tactin, añadir avidina a una concentración final de 100 mg / ml y girar durante 15 minutos a 4 ° C. <ol><li> Si la resina reticulada de la etapa 1.10 ha sido almacenado durante un período prolongado de tiempo a 4 ° C, recoger la resina por centrifugación a 400 xg FOR 1 min a 4 ° C, descartar el sobrenadante y lavar la resina con 1 ml de TBST. Repetir la centrifugación y etapas de lavado dos veces más, primero con TBST y después con TBS. Centrifugar el tubo a 400 xg durante 1 min a 4 ° C y ajustar hasta el volumen original mediante la eliminación de exceso de TBS. </li></ol></li><li> Resuspender la resina Strep-Tactin reticulado mediante agitación. Inmediatamente después, añadir 50 l de la suspensión de resina al tubo que contiene la muestra de proteína cebo usando una punta de pipeta de corte y girar durante otros 30 min a 4 ° C. </li><li> Mientras espera, establecer termomezclador a 55 ° C y precalentar 500 l de tampón de elución para el uso en el paso 3.1. </li><li> Centrifugar a 400 xg durante 1 min a 4 ° C. Eliminar el sobrenadante y lavar la resina en un aparato rotatorio durante 5 min a 4 ° C con 1 ml de pre-refrigerada tampón de lavado # 1. Repetir la centrifugación y etapas de lavado tres veces. En el último paso, resuspender la resina en 250 l de tampón de lavado # 2. </li><li> Tr<html> ansfer la suspensión de resina a una columna de giro fresco. Resuspender la resina que queda en el tubo en otros 250 l de tampón de lavado # 2 y transferir a la misma columna. </li><li> Se centrifuga a 400 g durante 3 min a 4 ° C, desechar el flujo a través y la transferencia de la columna a un tubo ml delfines nariz 2 fresco. Proceda inmediatamente a la etapa de elución a continuación. </li></ol> 3. La elución de los complejos de proteínas específicas <ol><li> Añadir 150 l de tampón de elución precalentado desde el paso 2.4 a la columna de centrifugación. </li><li> Incubar en termomezclador durante 5 min a 55 ° C, agitando a 1400 rpm. </li><li> Centrifugar a 1500 xg durante 1 min a temperatura ambiente. </li><li> Deseche la columna y almacenar las proteínas diana purificadas a ≤ -20 ° C. </li></ol><table border="0" cellpadding="0" cellspacing="0" width="598"><tbody><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> Tampón fosfato salino (PBS) </td></tr>0 &quot;&gt; <td height="20" style="height:20px;width:299px;"> Na 2 HPO 4 </td><td style="width:299px;"> 10 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> KH 2 PO 4 </td><td style="width:299px;"> 2 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> NaCl </td><td style="width:299px;"> 137 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> KCl </td><td style="width:299px;"> 2,7 mM </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> pH ajustado a 7,4 a menos que se indique lo contrario (de pH 8,0 en la sección 1.4) </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> Solución salina tamponada con Tris (TBS) </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> Tris-HCl, pH 7,4 </td><td style="width:299px;"> 50 mM </td></tr><tr height="20"><td hei<html> ght = estilo &quot;20&quot; = &quot;altura: 20px; ancho: 299px;&quot;&gt; NaCl </td><td style="width:299px;"> 150 mM </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> Tris amortiguada salina con Tween 20 (TBST) </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> Tris-HCl, pH 7,4 </td><td style="width:299px;"> 50 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> NaCl </td><td style="width:299px;"> 150 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> De Tween 20 </td><td style="width:299px;"> 0,1% (v / v) </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> El tampón de unión </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> Tris-HCl, pH 8,0 </td><td style="width:299px;"> 25 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> NaCl </td>x &quot;&gt; 550 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> NaF </td><td style="width:299px;"> 5 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> EDTA </td><td style="width:299px;"> 0,5 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> Glicerol </td><td style="width:299px;"> 10% (v / v) </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> PMSF </td><td style="width:299px;"> 0,1 mM </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> Tampón de lavado # 1 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> Tris-HCl, pH 8,0 </td><td style="width:299px;"> 25 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> NaCl </td><td style="width:299px;"> 500 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> NaF </<html> td&gt; <td style="width:299px;"> 5 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> EDTA </td><td style="width:299px;"> 0,4 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> Igepal CA-630 </td><td style="width:299px;"> 0,2% (v / v) </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> Glicerol </td><td style="width:299px;"> 5% (v / v) </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> PMSF </td><td style="width:299px;"> 0,1 mM </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> Tampón de lavado # 2 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> Tris-HCl, pH 8,0 </td><td style="width:299px;"> 25 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> NaCl </td><td style="width:299px;"> 150 mM </td></tr><tr height="20">2 &quot;height =&quot; &quot;style =&quot; 20 altura: 20px; ancho: 597px; &quot;&gt; Tampón de elución </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> Tris-HCl, pH 8,0 </td><td style="width:299px;"> 25 mM </td></tr><tr height="22"><td height="22" style="height:22px;width:299px;"> SDS </td><td style="width:299px;"> 1% (w / v) </td></tr></tbody></table> Tabla 1. Tampones utilizados en el presente estudio.

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No hay conflictos de interés declarado.

El protocolo anterior se puede utilizar para purificar cualquier proteína cebo de etiquetado de doble Strep de interés y sus complejos asociados en cualquier tampón adecuado que no contiene biotina o desnaturalizantes fuertes. En la versión actual del protocolo, de unión y lavado se llevan a cabo bajo condiciones relativamente estrictas en presencia de altas concentraciones de sal y detergente no iónico. Aunque esto da como resultado en menos de fondo, los complejos de proteínas frágiles pueden disociarse bajo e...

En los últimos años, la tecnología Strep-tag ha sido ampliamente utilizado en muchas áreas de la investigación biomédica, incluida la proteómica y la biología estructural. Esta tecnología de purificación de proteínas, que se basa en la fusión de proteínas recombinantes a un péptido Strep-tag corto, ha madurado con el advenimiento de matrices de afinidad que llevan Strep-Tactin, una variante modificada genéticamente de la estreptavidina con la mejora de la capacidad de unión al péptido. 1, 2 Las proteínas de fusión que contienen dos copias de Strep-tag II, designado twin-Strep-tag o SIII-tag, muestran una mayor afinidad por matrices Strep-Tactin que las que contienen sólo un único Strep-tag, asegurando la purificación más eficiente de las proteínas recombinantes y sus parejas de unión asociados. Sin embargo, la mayor afinidad de las proteínas marcadas con doble Strep a Strep-Tactin también tiene su lado negativo. Elución competitiva de estas proteínas con el exceso de biotina puede ser incompleta, lo que lleva a la disminución de la producción de proteína objetivo. Una mmineral eficiente alternativa es elución con SDS, pero conduce a contaminación de la muestra no deseado con Strep-Tactin liberado de la resina, por lo que el ensayo incompatible con el análisis proteómico. Este trabajo presenta una técnica para superar esta limitación por primera estabilizar el tetrámero unido a resina de Strep-Tactin por entrecruzamiento químico y luego utilizando SDS para eluir proteínas etiquetadas-twin-Strep y sus complejos asociados de la resina reticulada resultante. Por lo tanto, suficiente rendimiento de proteína se puede lograr sin contaminación de la muestra con Strep-Tactin, permitiendo de este modo su posterior análisis por espectrometría de masas. El método es adecuado para la purificación de cualquier proteína de fusión recombinante con una SIII-tag expuesto a la superficie 3 o twin-Strep-tag (secuencia de aminoácidos WSHPQFEK (GGGS) 3 WSHPQFEK y SAWSHPQFEK (GGGS) 2 GGSAWSHPQFEK, respectivamente). La proteína puede ser de origen animal, vegetal o bacteriana y puede ser aislado a partir de ya sea total de célulaslisado o fracción enriquecida orgánulo. Como un ejemplo, se describe aquí la purificación de una proteína-SIII etiquetado VPg-Pro de la patata de virus A (PVA) 4 de la fracción nuclear de plantas de Nicotiana benthamiana infectadas por el PVA. La fracción nuclear se aisló tal como se describe anteriormente 5, con las siguientes modificaciones: las células no fueron tratados con formaldehído, butirato de sodio fue sustituido en todos los tampones con fluoruro de sodio 5 mM, inhibidor de proteasa completo fue sustituido con PMSF, Triton X-100 en la extracción de concentración tampón # 2 se redujo a 0,3% (v / v) y el sedimento nuclear obtenido por centrifugación a través de colchón de sacarosa (tampón de extracción # 3) se resuspendió en 1,45 ml de tampón de unión enfriado previamente y se hace girar durante 1,5 horas a 4 ° C. El extracto nuclear resultante que contiene la proteína cebo-etiquetados SIII y complejos asociados (muestra cebo de proteína) se procesó de acuerdo con el protocolo descrito a continuación (véase la sección 2).

<table border="0" cellpadding="0" cellspacing="0" width="606">
	<tbody>
		<tr>
			<td>Bis(sulfosuccinimidyl) suberate (BS3), No-Weigh format, 8 x 2mg</td>
			<td>Pierce/Thermo Scientific</td>
			<td>21585</td>
			<td><a href="http://www.fishersci.com">www.fishersci.com CAUTION: Hazardous substance. Causes serious respiratory, skin and eye irritation. Wear protective gloves and eye protection.</a></td>
		</tr>
		<tr>
			<td>Strep-Tactin MacroPrep resin (50% suspension)</td>
			<td>IBA</td>
			<td>2-1505</td>
			<td><a href="http://www.iba-lifesciences.com/">www.iba-lifesciences.com</a></td>
		</tr>
		<tr>
			<td>Spin-X centrifuge tube filter, cellulose acetate membrane, pore size 0.45 &#956;m, non-sterile</td>
			<td>Costar (Corning)</td>
			<td>8163</td>
			<td><a href="http://www.corning.com/lifesciences/">www.corning.com/lifesciences/</a></td>
		</tr>
		<tr>
			<td>Dolphin-nose tubes</td>
			<td>Costar (Corning)</td>
			<td>3213</td>
			<td><a href="http://www.corning.com/lifesciences/">www.corning.com/lifesciences/</a></td>
		</tr>
		<tr>
			<td>Avidin&#160;</td>
			<td>IBA</td>
			<td>2-0204</td>
			<td><a href="http://www.iba-lifesciences.com/">www.iba-lifesciences.com</a></td>
		</tr>
	</tbody>
</table>

one-step purification of twin-strep-tagged proteins and their complexes on strep-tactin resin cross-linked with bis(sulfosuccinimidyl) suberate (bs3)

La purificación por afinidad de proteínas de fusión Strep-etiquetados en resinas que llevan una estreptavidina de ingeniería (Strep-Tactin) se ha convertido en un método ampliamente utilizado para el aislamiento de complejos de proteínas en condiciones fisiológicas. Las proteínas de fusión que contienen dos copias de Strep-tag II, designado doble-Strep-tag o SIII-etiqueta, tienen la ventaja de una mayor afinidad por Strep-Tactin en comparación con los que contienen sólo un único marcador Strep, permitiendo de este modo la purificación de proteínas más eficiente. Sin embargo, esta ventaja se ve compensada por el hecho de que la elución de las proteínas marcadas con doble Strep con biotina puede ser incompleta, lo que lleva a la recuperación baja en proteínas. La recuperación se puede mejorar drásticamente mediante el uso de elución desnaturalizante con dodecil sulfato de sodio (SDS), pero esto conduce a la contaminación de la muestra con Strep-Tactin liberado de la resina, por lo que el ensayo incompatible con el análisis proteómico de aguas abajo. Para superar esta limitación, se ha desarrollado un método por el cual tetrámero acoplado a la resina de Strep-Tactinprimero se estabiliza mediante la reticulación covalente con bis (sulfosuccinimidil) suberato (BS3) y la resina reticulada resultante se utiliza a continuación para purificar los complejos de proteína diana en una única etapa de purificación por lotes. Elución eficiente con SDS asegura una buena recuperación de proteína, mientras que la ausencia de contaminación de Strep-Tactin permite el análisis de proteínas aguas abajo por espectrometría de masas. Como una prueba de concepto, se describe aquí un protocolo para la purificación de la proteína viral SIII etiquetada VPg-Pro a partir de núcleos de N. infectados por el virus benthamiana plantas utilizando la resina de polimetacrilato de Strep-Tactin reticulado con BS3. El mismo protocolo se puede utilizar para purificar cualquier proteína marcada-Strep-gemelo de interés y caracterizar sus parejas de unión fisiológicos.

El procedimiento de purificación se ilustra esquemáticamente en la Figura 1, junto con una representación de los problemas asociados con otros métodos de purificación existentes. <img alt="Figura 1" fo:content-width="5.5in" src="/files/ftp_upload/51536/51536fig1highres.jpg" width="650" /> Figura 1. Representación esquemática del procedimiento de purificación. El flujo de trabajo incluye la estabilización de...

Watch this Scientific Journal Video about One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bissulfosuccinimidyl Suberate BS3 at JoVE.com

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bissulfosuccinimidyl Suberate BS3

Se describe un método para la purificación eficiente de proteínas de fusión doble-Strep-etiquetados y sus complejos específicos sobre estreptavidina modificada (Strep-Tactin) de resina covalentemente reticulado con bis (sulfosuccinimidil) suberato (BS3). El método tiene las ventajas de velocidad rápida, buena recuperación proteína diana y alta pureza, y es compatible con el análisis posterior por espectrometría de masas.

De un solo paso de purificación de proteínas etiquetadas-Twin-Strep y sus complejos de Strep-Tactin resina reticulado con Bis (sulfosuccinimidil) suberato (BS3)

Affinity purification of Strep-tagged fusion proteins on resins carrying an engineered streptavidin (Strep-Tactin) has become a widely used method for ...

one-step-purification-twin-strep-tagged-proteins-their-complexes-on

Research

JoVE Journal

Biology

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bis(sulfosuccinimidyl) Suberate (BS3)

18.6K vistas.  University of Helsinki.  Se describe un método para la purificación eficiente de proteínas de fusión doble-Strep-etiquetados y sus complejos específicos sobre estreptavidina modificada (Strep-Tactin) de resina covalentemente reticulado con bis (sulfosuccinimidil) suberato (BS3). El método tiene las ventajas de velocidad rápida, buena recuperación proteína diana y alta pureza, y es compatible con el análisis posterior por espectrometría de masas. 

Video: One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bissulfosuccinimidyl Suberate BS3

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A nucleoprotein (NP) into viral ribonulceoprotein particles (vRNPs). The influenza vRNPs are enclosed within the viral envelope. During cell entry, however, these vRNP complexes are released into the cytoplasm, where they gain access to the host nuclear transport machinery. In order to study the nuclear import of influenza vRNPs and the replication of the influenza genome, it is useful to work with isolated vRNPs so that other components of the virus do not interfere with these processes. Here, we describe a procedure to purify these vRNPs from the influenza A virus. The procedure starts with the disruption of the influenza A virion with detergents in order to release the vRNP complexes from the enveloped virion. The vRNPs are then separated from the other components of the influenza A virion on a 33-70% discontinuous glycerol gradient by velocity sedimentation. The fractions obtained from the glycerol gradient are then analyzed on via SDS-PAGE after staining with Coomassie blue. The peak fractions containing NP are then pooled together and concentrated by centrifugation. After concentration, the integrity of the vRNPs is verified by visualization of the vRNPs by transmission electron microscopy after negative staining. The glycerol gradient purification is a modification of that from Kemler et al. (1994)1, and the negative staining has been performed by Wu et al. (2007).2

The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza 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 ...

Investigación

Biología

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Identificación de los complejos de proteínas con la proteómica cuantitativa en S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

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 ...

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

Los estudios genéticos de proteínas humanas de reparación del ADN utilizando la levadura como un sistema modelo

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

One-step Metabolomics: Carbohydrates, Organic and Amino Acids Quantified in a Single Procedure

Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on tandem mass spectrometry and quantifies acylcarnitines as a screen for organic acidemias and also measures amino acids. All states also perform enzymatic testing for carbohydrate disorders such as galactosemia. Because the results can be non-specific, follow-up testing of positive results is required using a more definitive method. The present report describes the "urease" method of sample preparation for inborn error screening. Crystalline urease enzyme is used to remove urea from body fluids which permits most other water-soluble metabolites to be dehydrated and derivatized for gas chromatography in a single procedure. Dehydration by evaporation in a nitrogen stream is facilitated by adding acetonitrile and methylene chloride. Then, trimethylsilylation takes place in the presence of a unique catalyst, triethylammonium trifluoroacetate. Automated injection and chromatography is followed by macro-driven custom quantification of 192 metabolites and semi-quantification of every major component using specialized libraries of mass spectra of TMS derivatized biological compounds. The analysis may be performed on the widely-used Chemstation platform using the macros and libraries available from the author. In our laboratory, over 16,000 patient samples have been analyzed using the method with a diagnostic yield of about 17%--that is, 17% of the samples results reveal findings that should be acted upon by the ordering physician. Included in these are over 180 confirmed inborn errors, of which about 38% could not have been diagnosed using previous methods.

The urease method of sample preparation for GC/MS analysis of intermediary metabolites is presented by its inventor. The method allows one-step follow-up of newborn screening for inborn errors by tandem mass spectrometry by quantifying carbohydrates, organic and amino acids all in a single process.

De un solo paso metabolómica: Hidratos de carbono, ácidos orgánicos y aminoácidos cuantificados en un procedimiento único

Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on ...

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 ...

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 ...

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is considered a gentle dehydration method. To facilitate cell survival during drying, the cells are often formulated beforehand. The formulation forms a matrix that embeds the cells and protects them from various harmful stresses imposed on the cells during freezing and drying. We present here a general method to evaluate the survival rate of cells after freeze-drying and we illustrate it by comparing the results obtained with four different formulations: the disaccharide sucrose, the sucrose derived polymer Ficoll PM400, and the respective polysaccharides hydroxyethyl cellulose (HEC) and hydroxypropyl methyl cellulose (HPMC), on two strains of bacteria, P. putida KT2440 and A. chlorophenolicus A6. In this work we illustrate how to prepare formulations for freeze-drying and how to investigate the mechanisms of cell survival after rehydration by characterizing the formulation using of differential scanning calorimetry (DSC), surface tension measurements, X-ray analysis, and electron microscopy and relating those data to survival rates. The polymers were chosen to get a monomeric structure of the respective polysaccharide resembling sucrose to a varying degrees. Using this method setup we showed that polymers can support cell survival as effectively as disaccharides if certain physical properties of the formulation are controlled1.

Freeze-drying is often an easy and convenient way to obtain dry products of viable bacterial cells. An issue of the process is cell survival. We detail here a procedure to investigate how cell survival during freeze-drying is influenced by the properties of the formulation used.

Las formulaciones para la liofilización de las bacterias y su influencia en la supervivencia celular

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

De alto rendimiento de purificación de afinidad de proteínas marcadas con recombinantes

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Purification of Transcripts and Metabolites from Drosophila Heads

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

Purification of Transcripts and Metabolites from Drosophila Heads

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

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

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