Este estudio fue apoyado por un Premio al Mérito del Departamento de Asuntos de Veteranos de Estados Unidos, y un NIH conceder R01CA138528 a RW.

1 Preparación de construcciones de expresión <ol><li> Antes de comenzar el protocolo, PRMTs clones en vectores de expresión de mamífero con una etiqueta de epítopo N-terminal (Myc o HA), y el sustrato en marco con la glutatión-S-transferasa (GST) para la expresión de proteínas de alto nivel y purificación a partir de células bacterianas lisados, usando técnicas de biología molecular estándar. </li></ol> 2. Purificación de PRMTs <ol><li> Utilice 100 mm cultura platos para mantener las células epiteliales bronquiales humanas (Beas2B). Pasaje de las células cada 3 días y no dejarlos crecer hasta confluencia. </li><li> En el día antes de la transfección, las semillas 7,5 x 10 5 células en medio de cultivo de 10 ml en una placa de cultivo de 100 mm. Agitar para dispersar uniformemente las células, y se incuba a 37 ° C en un incubador de CO2 humidificado 5%. </li><li> Al día siguiente, aspirar los medios de comunicación y añadir 5 ml de medio de cultivo sin suero y antibióticos. Agitar y aspirar el medio. Repita este paso unomás tiempo y, finalmente, añadir 5 ml de medio libre de suero / antibiótico y proceder a la transfección. </li><li> Diluir 6 g de ADN plásmido en 750 l de medio libre de suero en un tubo de centrífuga de plástico de 1,5 ml estéril. </li><li> En otro tubo de centrífuga de plástico de 1,5 ml, diluir 30 l de reactivo de transfección en 750 l de medio libre de suero. </li><li> Transferir la solución diluida reactivo de transfección (paso 2,5) al tubo que contiene el ADN diluido (paso 2.4). Mezclar suavemente con la pipeta hacia arriba y abajo varias veces. </li><li> Incubar la mezcla de transfección a temperatura ambiente durante 15 min. </li><li> Después de 15 minutos, la mezcla de transfección pipetear suavemente sobre las células, remolino para mezclar uniformemente, y se incuba a 37 ° C en un incubador de CO2 humidificado 5%. </li><li> Después de 3 horas, aspirar el medio que contiene mezclas de transfección de las células. Añadir medio de cultivo celular fresco suplementado con suero y antibióticos. </li><li> Se incuba a 37 ° C en 5% de CO 2 humidificadoincubadora durante 48 horas. </li><li> Después de 48 h, la lisis de las células en 1 ml de tampón de lisis (1x solución salina tamponada con fosfato, 1% Nonidet P-40, 0,5% de desoxicolato de sodio, 0,1% de dodecil sulfato de sodio, y 1 mg / ml phenlymethyl sulfonil fluoruro). Recoger los lisados ​​en un tubo de centrífuga de plástico con un levantador de células e incubar los lisados ​​en hielo durante 30 min. </li><li> Centrifugar los lisados ​​a 15.000 xg durante 10 min en una centrífuga de sobremesa para borrar los lisados ​​de restos celulares. [* Se recomienda confirmar la expresión de HA-PRMT1 través inmunotransferencia de una parte alícuota de los lisados ​​con anticuerpos anti-HA, antes de proceder a la purificación de las enzimas para ensayo de metilación.] </li><li> Para la purificación de PRMTs, incubar 3 mg de lisados ​​celulares con 30 l de 50:50 suspensión de matriz de afinidad anti-HA en tampón fosfato salino (PBS), y rotar durante la noche a 4 ° C en un rotador de tubo. </li><li> Al día siguiente, girar los tubos a 14.000 xg en una centrífuga de sobremesa durante 2 minutos para recogerlas perlas. Descartar el sobrenadante y lavar las perlas 3 veces con 1 ml de tampón RIPA [Tris 20 mM (pH 8,0), NaCl 150 mM, EDTA 5 mM y 1% de Triton-X-100] en cada lavado seguido de centrifugación a 14.000 xg durante 2 min. Después del tercer lavado, lavar las perlas una vez con 100 l de tampón de metilación (50 mM Tris pH 8,5, 20 mM KCl, 10 mM de MgCl 2, 1 mM β-mercaptoetanol, sacarosa 100 mM). Después de la centrifugación, eliminar el sobrenadante. Las perlas que contienen los PRMTs inmovilizados están listos para ser utilizado en el ensayo de metilación. [* Se recomienda utilizar los PRMTs inmovilizados inmediatamente en el ensayo de metilación.] </li></ol> 3. Purificación de Sustrato <ol><li> Inocular una sola colonia de GST-G3BP1 transformó BL21 en 5 ml de medio Luria-Bertani (LB) que contenían 100 mg / ml de ampicilina. Crecer los cultivos durante la noche a 37 ° C con agitación a 250 rpm. </li><li> Al día siguiente, transferir el cultivo durante la noche a 50 ml de medio LB fresco y continue crezca a un OD 600 de 0,5-0,7 (tarda aproximadamente 2 horas). </li><li> Cuando el OD 600 alcanza 0,5-0,7, añadir isopropil-β-D-tiogalactósido (IPTG) a una concentración final de 1 mM y continuar a la cultura de 4 horas adicionales a 37 ° C con agitación a 250 rpm. </li><li> Recoger las células por centrifugación a 1500 xg durante 10 minutos, y el proceso de la pastilla para la purificación GST o almacenar a -80 ° C. </li><li> Vuelva a suspender el sedimento bacteriano en 5 ml de tampón de lisis (PBS 1x con inhibidores de proteasa, 100 g / ml de lisozima, y ​​0,1% de Triton-X-100) y se incuban los lisados ​​a 37 ° C durante 30 min. En este momento los lisados ​​aparecerá turbia y viscosa debido a la liberación de ADN genómico bacteriano. </li><li> Cortar el ADN genómico por tratamiento con ultrasonidos (10 pulsos de 1 seg cada uno con una amplitud de 30%). </li><li> Borrar los lisados ​​por centrifugación a 15.000 xg durante 10 min y transferir el sobrenadante transparente a un tubo de 15 ml. Si el sobrenadante no está claro repetir el centrifugación proceso una vez más. </li><li> Mientras tanto, lavar las perlas de sefarosa-GST dos veces con PBS enfriado con hielo y hacer una suspensión 50:50 de GST-sefarosa en PBS. Añadir 200 l de la suspensión GST-sefarosa al lisado celular bacteriana despejado y rotar a 4 ° C durante 1 hr. </li><li> Después de 1 hora, centrifugar los tubos a 500 xg durante 3 minutos para recoger las perlas. Lavar las perlas 3 veces con PBS frío hielo. </li><li> Después del lavado final, resuspender las perlas en 100 l de tampón de elución (50 mM Tris pH 8,0, NaCl 150 mM, glutatión 30 mM) y girar a 4 ° C durante 10 min. </li><li> Centrifugar los tubos a 15.000 xg durante 2 min y transferir el sobrenadante que contiene la proteína purificada en otro tubo de centrífuga de plástico. Llevar a cabo la electroforesis en gel de poliacrilamida SDS (SDS-PAGE) análisis sobre 5 l de la proteína purificada junto con cantidades conocidas de albúmina de suero bovino (BSA) para estimar la concentración de la proteína purificada. </li></ol> 4. metilación Ensayo NOTA: Realice los pasos siguientes en un área separada designada para el trabajo de radioisótopos y de acuerdo con los protocolos de la institución en la realización de experimentos con isótopos radiactivos. <ol><li> Añadir 2 g de GST-sustrato purificada y una mezcla maestra que contiene tampón de metilación 1x (50 mM Tris pH 8,5, 20 mM KCl, 10 mM de MgCl 2, 1 mM β-mercaptoetanol, sacarosa 100 mM), 1 Ci de S-adenosil- L-[metil-3H] metionina, y agua hasta un volumen final de 10 l a los PRMTs inmovilizados (paso 2,13) ​​y se incuba la reacción a 30 ° C durante 1 hr. </li><li> Después de 1 hora, detener la reacción mediante la adición de 10 l de tampón de muestra 2x SDS. Después de la desnaturalización de las muestras a 95 ° C durante 10 min, separar las muestras en un gel de SDS-PAGE. </li><li> Más tarde, transferir las proteínas separadas del gel de SDS sobre membrana de nitrocelulosa mediante el uso de una transferencia electroforética semi-seco. </li><li> Después de la transferencia, cubrir la membrana con unutofluor (un intensificador de imagen autorradiográfica), y el rock lento durante 2 horas a temperatura ambiente. Después de 2 horas, verter la autofluor y conservar para su uso posterior. [* Etiquetar el tubo como material radiactivo] </li><li> Aire rápidamente se seque la membrana en un papel de filtro, sellar en una bolsa de plástico y exponer la membrana a una película de rayos X a -20 ° C. Tiempo de exposición suficiente oscila entre 5-30 días. </li></ol> 5. Confirmación de la expresión PRMT y la igualdad de carga de Sustrato <ol><li> Después de lograr exposiciones suficientes, transferir la membrana a una caja de plástico que contiene tampón de bloqueo [Tris-buffer salino con Tween-20 (TBST + leche descremada 3%)] y el rock suave durante 1 hora. </li><li> Deseche el tampón de bloqueo como residuos radiactivos. Incubar la membrana con anticuerpo anti-HA (dilución 1: 1.000) en TBST durante la noche a 4 ° C, con suave balanceo. </li><li> Lavar la membrana con TBST 3x e incubar la membrana con marcado con HRP anti-ratón anticuerpo secundario(1: 5000 dilución) en TBST a temperatura ambiente durante 1 h con agitación suave. </li><li> Después de 1 hora, lavar la membrana con TBST 3x, y realizar la detección de quimioluminiscencia de proteínas con un sustrato de HRP. </li><li> Después de la detección de los PRMTs HA-etiquetados, incubar la membrana con solución de tinción Ponseau-S durante 5 min a temperatura ambiente. Lavar rápidamente las 3x membrana con agua para detectar los sustratos GST-etiquetados. Escanear la imagen. </li></ol>

<ol>
	<li>Paik, W. K., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+methylase+I.+Purification+and+properties+of+the+enzyme.">Protein methylase I. Purification and properties of the enzyme.</a> J Biol Chem. 243, 2108-2114 (1968).</li><li>Lin, W. J., Gary, J. D., Yang, M. C., Clarke, S., Herschman, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mammalian+immediate-early+TIS21+protein+and+the+leukemia-associated+BTG1+protein+interact+with+a+protein-arginine+N-methyltransferase.">The mammalian immediate-early TIS21 protein and the leukemia-associated BTG1 protein interact with a protein-arginine N-methyltransferase.</a> J Biol Chem. 271, 15034-15044 (1996).</li><li>Boffa, L. C., Karn, J., Vidali, G., Allfrey, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+NG,+NG,-dimethylarginine+in+nuclear+protein+fractions.">Distribution of NG, NG,-dimethylarginine in nuclear protein fractions.</a> Biochem Biophys Res Commun. 74, 969-976 (1977).</li><li>Bedford, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine+methylation+at+a+glance.">Arginine methylation at a glance.</a> J Cell Sci. 120, 4243-4246 (2007).</li><li>Lee, Y. H., Stallcup, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minireview:+protein+arginine+methylation+of+nonhistone+proteins+in+transcriptional+regulation.">Minireview: protein arginine methylation of nonhistone proteins in transcriptional regulation.</a> Mol Endocrinol. 23, 425-433 (2009).</li><li>Bedford, M. T., Clarke, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+arginine+methylation+in+mammals:+who,+what,+and+why.">Protein arginine methylation in mammals: who, what, and why.</a> Mol Cell. 33, 1-13 (2009).</li><li>Bikkavilli, R. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dishevelled3+is+a+novel+arginine+methyl+transferase+substrate.">Dishevelled3 is a novel arginine methyl transferase substrate.</a> Scientific reports. 2, 805 (2012).</li><li>Bikkavilli, R. K., Malbon, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine+methylation+of+G3BP1+in+response+to+Wnt3a+regulates+{beta}-catenin+mRNA.">Arginine methylation of G3BP1 in response to Wnt3a regulates {beta}-catenin mRNA.</a> J Cell Sci. 124, 2310-2320 (2011).</li><li>Bikkavilli, R. K., Malbon, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt3a-stimulated+LRP6+phosphorylation+is+dependent+upon+arginine+methylation+of+G3BP2.">Wnt3a-stimulated LRP6 phosphorylation is dependent upon arginine methylation of G3BP2.</a> J Cell Sci. , (2012).</li><li>Tini, M., Naeem, H., Torchia, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+analysis+of+arginine+methylation+in+transcription.">Biochemical analysis of arginine methylation in transcription.</a> Methods Mol Biol. 523, 235-247 (2009).</li><li>Lee, J., Cheng, D., Bedford, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+in+protein+methylation.">Techniques in protein methylation.</a> Methods Mol Biol. 284, 195-208 (2004).</li><li>Cheng, D., Vemulapalli, V., Bedford, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+applied+to+the+study+of+protein+arginine+methylation.">Methods applied to the study of protein arginine methylation.</a> Methods Enzymol. 512, 71-92 (2012).</li><li>Zurita-Lopez, C. I., Sandberg, T., Kelly, R., Clarke, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+protein+arginine+methyltransferase+7+(PRMT7)+is+a+type+III+enzyme+forming+omega-NG-monomethylated+arginine+residues.">Human protein arginine methyltransferase 7 (PRMT7) is a type III enzyme forming omega-NG-monomethylated arginine residues.</a> J Biol Chem. 287, 7859-7870 (2012).</li></ol>

Los autores no tienen nada que revelar.

El protocolo descrito en el presente documento se utiliza rutinariamente para establecer el estado de metilación de los sustratos PRMT identificados. Este ensayo robusto y consistente también proporcionará evidencia definitiva de la especificidad de PRMTs para sus sustratos. Los componentes clave para el éxito de este ensayo son: 1. Expresión de PRMTs en células de mamífero, 2. Actividad de PRMTs purificados, 3. Expresión y purificación de sustrato, y 4. transferencia Western completa de las proteínas a la mem...

Metilación de proteínas fue descrita por primera vez en 1968 1. No fue sino hasta la primera clonación de PRMT1 en 1996, que los investigadores comenzaron a apreciar la importancia de esta modificación después de la traducción 2. Curiosamente, alrededor del 2% de los residuos de arginina en las proteínas de extractos nucleares se metilado 3, lo que indica la abundancia de esta modificación. La arginina es un aminoácido cargado positivamente con una cadena lateral básica y el nitrógeno / s dentro de las cadenas laterales de arginina puede ser modificado después de la traducción a través de la adición de un grupo metilo, un proceso conocido como arginina metilación 4-6. Metilación de arginina es catalizada por una clase de enzimas viz., Transferasas proteína arginina metil (PRMTs). Argininas pueden o bien ser monometilado o dimetilado y el segundo pueden ser simétricos o asimétricos, dependiendo del tipo de PRMTs que catalizan el proceso de 4-6. Las proteínas que muestran argininamotivos ricos e-glicina son objetivos potenciales para la catálisis PRMT mediada. PRMT metilación mediada de sustratos se ha demostrado que modulan la interacción proteína-proteína, interacciones proteína-ácido nucleico, la función de proteínas, la expresión génica, y / o de señalización celular, todos los cuales son críticos para la homeostasis celular normal 7-9. Con el fin de entender el papel biológico de la proteína metilación de arginina, se requieren ensayos precisos, eficientes y reproducibles para establecer el estado de metilación de los sustratos PRMT identificados. En ensayo de metilación in vitro, que evalúa las capacidades de PRMTs purificadas para catalizar la metilación de sus sustratos, es un ensayo bien aceptado para el estudio de la metilación de arginina 10-12. El éxito general de este ensayo depende en gran medida de la actividad de los PRMTs purificados. PRMTs pueden ser expresadas y purificadas a partir de bacterias o células de mamífero, 10,11. Este ensayo de metilación in vitro, como detailed en la sección de protocolo, se basa en un método descrito originalmente por Tini y sus colegas 10. En este protocolo, se muestra en detalle los pasos necesarios para la expresión y purificación de PRMT1 en células de mamíferos. La capacidad de la PRMT1 purificada para catalizar la metilación en Ras GTPasa-activación de la proteína de unión a la proteína 1 (G3BP1), un sustrato conocido PRMT 8, más tarde fue evaluada en presencia de S-adenosil-L-[metil-3H] metionina como el donante de metilo. Utilizando este ensayo, podemos definir con fiabilidad las habilidades de los PRMTs a la novela metilato o sustratos conocidos, que es un paso principal en el estudio de la metilación de arginina de proteínas.

<table border="0" cellpadding="0" cellspacing="0" width="673">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">Name of Material/ Equipment</td>
			<td style="width:136px;">Company</td>
			<td style="width:111px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">S-adenosyl-L-[methyl-3H] methionine</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ecoscint ultra&#160;</td>
			<td>National Diagnostics</td>
			<td>LS-270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bottle top dispenser</td>
			<td>National Diagnostics</td>
			<td>LS-900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AutoFluor</td>
			<td>National Diagnostics</td>
			<td>LS-315</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 ml Scintillation Vials</td>
			<td>National Diagnostics</td>
			<td>SKU:SVC-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">GST-sepharose</td>
			<td style="width:136px;">GE Life Sciences</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">L-Glutathione Reduced</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:111px;">G4251</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Lipofectamine</td>
			<td style="width:136px;">Invitrogen</td>
			<td style="width:111px;"></td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Beas2B</td>
			<td style="width:136px;">ATCC</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Optimem</td>
			<td style="width:136px;">Invitrogen</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">NP-40</td>
			<td style="width:136px;">US-Biologicals</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">SDS</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Sodium deoxycholate</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">PMSF</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">anti-HA affinity matrix</td>
			<td style="width:136px;">Roche</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Tris</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Nacl</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Bio-rad gel doc</td>
			<td style="width:136px;">Bio Rad</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">anti-HA antibody</td>
			<td style="width:136px;">roche</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Ponseau S</td>
			<td style="width:136px;">Fisher Scientific</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro methylation assay to study protein arginine methylation

Proteína arginina metilación es una de las más abundantes modificaciones post-traduccionales en el núcleo. Proteína arginina metilación se puede identificar y / o determina a través de enfoques proteómicos, y / o inmunotransferencia con anticuerpos específicos metil-arginina. Sin embargo, estas técnicas a veces pueden ser engañosas ya menudo proporcionan resultados positivos falsos. Más importante, estas técnicas no pueden proporcionar evidencia directa en apoyo de la especificidad de sustrato PRMT. Los ensayos in vitro de metilación, por otro lado, son ensayos bioquímicos útiles, que son sensibles, y consistentemente revelan si las proteínas identificadas son de hecho los sustratos PRMT. Un ensayo típico de metilación in vitro incluye, PRMTs activos purificados, sustrato purificado y marcado con un radioisótopo metil donante (S-adenosil-L-[metil-3H] metionina). Aquí se describe un protocolo de paso a paso para aislar PRMT1 catalíticamente activo, un miembro de la familia PRMT doquier expresó. El metransferasa actividades Thyl del PRMT1 purificada más tarde fueron probados en Ras-proteína activadora de GTPasa proteína de unión 1 (G3BP1), un sustrato PRMT conocido, en presencia de S-adenosil-L-[metil-3H] metionina como donante de metilo. Este protocolo se puede emplear no sólo para establecer el estado de metilación de sustratos fisiológicos PRMT1 nuevos, sino también para la comprensión del mecanismo básico de la metilación de arginina de proteínas.

<html> Las habilidades de HA-PRMT1 de metilato de GST-G3BP1 se determinaron por un ensayo in vitro de metilación en. PRMT1 marcada con HA purificada a partir de lisados ​​de células Beas2B se empleó en una reacción de metilación que contiene GST-G3BP1, y 1 Ci de S-adenosil-L-[metil-3H] metionina, como un donante de metilo. PRMT1 podría catalizar eficazmente la metilación de GST-G3BP1 (Figura 1, panel superior). Reacciones de metilación utilizando menús desplegabl...

Watch this Scientific Journal Video about In vitro Methylation Assay to Study Protein Arginine Methylation at JoVE.com

In vitro Methylation Assay to Study Protein Arginine Methylation

Proteína arginina metilación, catalizada por una clase de enzimas viz., La proteína arginina metil transferasas (PRMTs), es el proceso de adición enzimática de grupo (s) metilo para argininas dentro de las proteínas. El ensayo de metilación in vitro es la herramienta más fiable para evaluar el estado de metilación de sustratos PRMT conocidos o nuevos.

In vitro metilación de ensayo para estudiar la proteína arginina metilación

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

in-vitro-methylation-assay-to-study-protein-arginine-methylation

Research

JoVE Journal

Biology

In vitro Methylation Assay to Study Protein Arginine Methylation

15.1K vistas.  University of Illinois at Chicago.  Proteína arginina metilación, catalizada por una clase de enzimas viz., La proteína arginina metil transferasas (PRMTs), es el proceso de adición enzimática de grupo (s) metilo para argininas dentro de las proteínas. El ensayo de metilación in vitro es la herramienta más fiable para evaluar el estado de metilación de sustratos PRMT conocidos o nuevos. 

Video: In vitro Methylation Assay to Study Protein Arginine Methylation

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Prueba de sensibilidad visual a la velocidad y dirección del movimiento en lagartos

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Investigación

Biología

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA methyltransferases is viable, thus providing a system to study how loss of DNA methylation in a genome affects growth and development. Imprinting refers to differential expression of maternal and paternal alleles and plays an important role in reproduction development in both mammal and plants. DNA methylation is critical for determining whether the maternal or paternal alleles of an imprinted gene is expressed or silenced. In flowering plants, there is a double fertilization event in reproduction: one sperm cell fertilizes the egg cell to form embryo and a second sperm fuses with the central cell to give rise to endosperm. Endosperm is the tissue where imprinting occurs in plants. MEDEA, a SET domain Polycomb group gene, and FWA, a transcription factor regulating flowering, are the first two genes shown to be imprinted in endosperm and their expression is controlled by DNA methylation and demethylation in plants. In order to determine imprinting status of a gene and methylation pattern in endosperm, we need to be able to isolate endosperm first. Since seed is tiny in Arabidopsis, it remains challenging to isolate Arabidopsis endosperm and examine its methylation. In this video protocol, we report how to conduct a genetic cross, to isolate endosperm tissue from seeds, and to determine the methylation status by bisulfite sequencing.

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Imprinting is a phenomenon in plant and mammal reproduction. DNA methylation plays an important role in mechanisms of imprinting. Isolating endosperm and determining methylation status of imprinted genes in Arabidopsis can be difficult. In this protocol, we describe how to isolate endosperm and determine methylation by bisulfite sequencing.

Determinación de la metilación del ADN de los genes impresos en Arabidopsis Endospermo

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

De proteínas de membrana de superposición de ensayo: Un protocolo de prueba de interacción entre las proteínas soluble e insoluble In vitro</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

La metilación del ADN: Bisulfito de modificación y análisis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

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

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

Una afinidad líquido fase de captura de ensayo utilizando partículas magnéticas para estudiar la interacción proteína-proteína: el ejemplo de poliomyelits-Nanobodies

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

Oxidative stress is an unavoidable byproduct of aerobic life. Molecular oxygen is essential for terrestrial metabolism, but it also takes part in many damaging reactions within living organisms. The combination of aerobic metabolism and iron, which is another vital compound for life, is enough to produce radicals through Fenton chemistry and degrade cellular components. DNA degradation is arguably the most damaging process involving intracellular radicals, as DNA repair is far from trivial. The assay presented in this article offers a quantitative technique to measure and visualize the effect of molecules and enzymes on radical-mediated DNA damage.
The DNA protection assay is a simple, quick, and robust tool for the in vitro characterization of the protective properties of proteins or chemicals. It involves exposing DNA to a damaging oxidative reaction and adding varying concentrations of the compound of interest. The reduction or increase of DNA damage as a function of compound concentration is then visualized using gel electrophoresis. In this article we demonstrate the technique of the DNA protection assay by measuring the protective properties of the DNA-binding protein from starved cells (Dps). Dps is a mini-ferritin that is utilized by more than 300 bacterial species to powerfully combat environmental stressors. Here we present the Dps purification protocol and the optimized assay conditions for evaluating DNA protection by Dps.

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

The DNA-binding protein from starved cells (Dps) plays a crucial role in combating bacterial stress. This article discusses the purification of E. coli Dps and the protocol for an in vitro assay demonstrating Dps-mediated protection of DNA from degradation by reactive oxygen species.

La aplicación de un<em&gt; In vitro</em&gt; DNA ensayo de protección de visualizar Estrés Mediación propiedades de la proteína Dps

Oxidative stress is an unavoidable byproduct of  aerobic life.  Molecular oxygen is  essential for terrestrial metabolism, but it also takes part in many ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

Ensayo de formación de tubos de células endoteliales para la<em&gt; In Vitro</em&gt; Estudio de la angiogénesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

Enhanced reducido Representación bisulfito secuenciación para la Evaluación de la metilación del ADN en la Resolución Base Par

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.

La correlación de Gene-específica de ADN de metilación cambios con la expresión y actividad transcripcional de astrocíticos<em&gt; KCNJ10</em&gt; (Kir4.1)