Este estudo foi apoiado por um Prémio de Mérito do Departamento de Assuntos de Veteranos dos Estados Unidos, e um NIH conceder R01CA138528 a RW.

1: Preparação de construções de expressão <ol><li> Antes de se iniciar o protocolo, PRMTs clones em vectores de expressão de mamíferos com um epitopo tag N-terminal (Myc ou HA), e o substrato em-quadro com a glutationa-S-transferase (GST) para a expressão de proteínas de nível elevado e purificação a partir de células bacterianas Os lisados, utilizando técnicas de biologia molecular padrão. </li></ol> 2 Purificação de PRMTs <ol><li> Utilizar placas de 100 mm de cultura para manter as células epiteliais brônquicas humanas (Beas2B). Passagem das células a cada três dias e não deixá-los crescer até confluência. </li><li> No dia antes da transfecção, 7,5 x 10 semear 5 células em 10 ml de meio de cultura de uma cultura de 100 mm de prato. Redemoinho para dispersar uniformemente as células, e incuba-se a 37 ° C numa atmosfera de 5% de CO 2 humidificado incubadora. </li><li> No dia seguinte, aspirar a mídia e adicionar 5 ml de meio de cultura sem soro e antibióticos. Agitar e aspire o meio. Repita este passo ummais tempo e, finalmente, adicionar 5 ml de soro / meio sem antibiótico e prossiga para transfecção. </li><li> Dilui-se 6 ug de ADN de plasmídeo em 750 ul de meio isento de soro em um tubo de 1,5 ml de centrífuga de plástico estéril. </li><li> Em outro tubo de 1,5 ml de centrífuga de plástico, diluir 30 ul de reagente de transfecção em 750 ul de meio isento de soro. </li><li> Transferir a solução de reagente de transfecção diluída (passo 2.5), para o tubo contendo ADN diluído (passo 2.4). Misture com cuidado pipetando para cima e para baixo várias vezes. </li><li> Incubar a mistura de transfecção à temperatura ambiente durante 15 min. </li><li> Após 15 min, a mistura de transfecção pipetar suavemente sobre as células, redemoinho para misturar uniformemente, e incubar a 37 ° C numa atmosfera de 5% de CO 2 humidificado incubadora. </li><li> Depois de 3 horas, aspirar o meio contendo misturas de transfecção das células. Adicionar meio de cultura celular fresco suplementado com soro e antibióticos. </li><li> Incubar a 37 ° C numa atmosfera de 5% de CO 2 humidificadoincubadora durante 48 horas. </li><li> Após 48 horas, lisar as células em 1 ml de tampão de lise (1 x solução salina tamponada com fosfato, 1% de Nonidet P-40, 0,5% desoxicolato de sódio, 0,1% de dodecil sulfato de sódio, e 1 ug / ml de fluoreto de sulfonilo phenlymethyl). Recolher os lisados ​​para um tubo de centrífuga de plástico com um tirante de células e incuba-se os lisados ​​em gelo durante 30 min. </li><li> Centrifugar os lisados ​​a 15000 xg durante 10 min numa centrífuga de bancada para limpar os lisados ​​a partir de detritos de células. [* É altamente recomendável para confirmar a expressão da HA-PRMT1 via immunoblotting de uma alíquota de lisados ​​com anticorpos anti-HA, antes de prosseguir para a purificação das enzimas, para ensaio de metilação.] </li><li> Para a purificação de PRMTs, incubar 3 mg de lisados ​​de células com 30 ul de suspensão de 50:50 anti-HA matriz de afinidade em tampão fosfato salino (PBS), e rodar durante a noite a 4 ° C em um tubo de rotor. </li><li> No dia seguinte, girar os tubos a 14.000 xg em uma centrífuga de bancada para 2 min para recolheros grânulos. Descartar o sobrenadante e lava-se as pérolas de 3x com 1 ml de tampão RIPA [Tris 20 mM (pH 8,0), NaCl 150 mM, EDTA 5 mM e 1% de Triton-X-100], em cada lavagem foi seguido por centrifugação a 14.000 xg durante 2 min. Após a terceira lavagem, lavagem das esferas uma vez com 100 ul de tampão de metilação (50 mM Tris pH 8,5, KCl 20 mM, 10 mM de MgCl2, 1 mM de β-mercaptoetanol, 100 mM de sacarose). Após a centrifugação, remove-se o sobrenadante. As contas contendo os PRMTs imobilizados está pronto para ser utilizado no ensaio de metilação. [* É altamente recomendável usar as PRMTs imobilizados imediatamente no ensaio de metilação.] </li></ol> 3 Purificação de Substrato <ol><li> Inocule uma colónia única de GST-G3BP1 BL21 transformada em 5 ml de meio de Luria-Bertani (LB) contendo 100 ug / ml de ampicilina. Crescer as culturas durante a noite a 37 ° C com agitação a 250 rpm. </li><li> No dia seguinte, a transferência da cultura durante a noite para 50 ml de meio fresco LB e contInue a crescer a um OD 600 de 0,5-0,7 (Demora cerca de 2 horas). </li><li> Quando a DO600 atinge 0,5-0,7, adicionar isopropil-β-D-tiogalactósido (IPTG) até uma concentração final de 1 mM e continuar a cultura durante 4 horas adicionais a 37 ° C com agitação a 250 rpm. </li><li> Colher as células por centrifugação a 1500 xg durante 10 min, e processar o pellet para a purificação de GST ou armazenar a -80 ° C. </li><li> Re-suspender o sedimento de bactérias em 5 ml de tampão de lise (PBS 1x com inibidores de protease, 100 ug / ml de lisozima, e 0,1% de Triton-X-100) e incuba-se os lisados ​​a 37 ° C durante 30 min. Nesta altura, os lisados ​​aparece turvo e viscoso, devido à libertação de ADN genómico bacteriano. </li><li> Cisalhamento do ADN genómico por ultra-sons (10 impulsos de 1 segundo cada um com uma amplitude de 30%). </li><li> Limpar os lisados ​​por centrifugação a 15.000 xg durante 10 minutos e transferir o sobrenadante límpido para um tubo de 15 ml. Se o sobrenadante é claro não repetir o centrifugação processo mais uma vez. </li><li> Enquanto isso, lavar as esferas de GST-Sepharose duas vezes com PBS gelado e se obter uma suspensão de GST-Sepharose em PBS de 50:50. Adicionar 200 ul da suspensão de GST-Sepharose para o ligado celular bacteriana foi afastada e rodam a 4 ° C durante 1 hora. </li><li> Depois de 1 hora, centrifugar os tubos a 500 xg por 3 min para recolher as esferas. Lavam-se as esferas de 3x com PBS gelado. </li><li> Após a lavagem final, voltar a suspender as pérolas em 100 ul de tampão de eluição (50 mM Tris pH 8,0, NaCl 150 mM, glutationa 30 mM) e rodar a 4 ° C durante 10 min. </li><li> Centrifugar os tubos a 15.000 xg durante 2 minutos e transfere-se o sobrenadante contendo a proteína purificada num outro tubo de centrífuga de plástico. Efectuar a electroforese de gel de poliacrilamida SDS (SDS-PAGE) em 5 ul de análise da proteína purificada, juntamente com quantidades conhecidas de albumina de soro bovino (BSA) para estimar a concentração de proteína purificada. </li></ol> 4. metilação Assay Nota: Execute seguintes passos em uma área separada designada para o trabalho de radioisótopos e de acordo com os protocolos da instituição sobre a realização de experimentos com isótopos radioativos. <ol><li> Adicionar 2 mg de substrato purificada GST-e uma mistura mestre contendo tampão de metilação 1x (50 mM Tris pH 8,5, KCl 20 mM, 10 mM de MgCl2, 1 mM de β-mercaptoetanol, 100 mM de sacarose), 1 uCi de S-adenosil- L-[metil-3H] metionina, e água até um volume final de 10 ul às PRMTs imobilizadas passo (2.13) e incuba-se a reacção a 30 ° C durante 1 hora. </li><li> Após 1 h, parar a reacção por adição de 10 ul de tampão de amostra 2x SDS. Após a desnaturação das amostras a 95 ° C durante 10 min, separar as amostras sobre um gel de SDS-PAGE. </li><li> Mais tarde, transferir as proteínas separadas a partir do gel de SDS para uma membrana de nitrocelulose usando uma transferência electroforética semi-seca. </li><li> Após a transferência, a membrana de cobertura com umautofluor (intensificador de imagem autorradiográfica), e agitar suavemente durante 2 horas à temperatura ambiente. Após 2 horas, despeje o autofluor e guarde para uso posterior. [* Rotular o tubo de material radioativo] </li><li> Ar rapidamente secar a membrana sobre um papel de filtro, selá-lo em um saco de plástico e expor a membrana a um filme de raios-X a -20 ° C. Tempo de exposição suficiente varia entre 5-30 dias. </li></ol> 5. Confirmação de Expressão PRMT e carga igual de substrato <ol><li> Depois de atingir posições suficientes, transferir a membrana a uma caixa de plástico que contém um tampão de bloqueio [solução salina tamponada com Tris com Tween-20 (TBST + 3% de leite magro)] e agitar suavemente durante 1 hora. </li><li> Descarte o tampão de bloqueio como resíduos radioactivos. Incubar a membrana com anticorpo anti-HA (1: 1000 de diluição) em TBST durante a noite a 4 ° C, com balanço suave. </li><li> Lava-se a membrana 3 x com TBST e incubar a membrana com um anticorpo secundário marcado com HRP anti-rato(1: 5000 de diluição) em TBST à temperatura ambiente durante 1 hora com agitação suave. </li><li> Após 1 h, lavar a membrana com TBST 3x, e realizar a detecção de proteínas chemiluminiscence com um substrato HRP. </li><li> Após a detecção dos PRMTs marcadas com HA, incubar a membrana com uma solução de coloração Ponseau-S, durante 5 minutos à temperatura ambiente. Lavar rapidamente as membranas 3x com água para detectar os substratos etiquetados com GST. Digitalizar a imagem. </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>

Os autores não têm nada a revelar.

O protocolo aqui descrito, é rotineiramente utilizada para estabelecer o estado de metilação dos substratos PRMT identificados. Este ensaio robusto e consistente também irá fornecer provas definitivas sobre a especificidade da PRMTs para os seus substratos. Os componentes essenciais para o sucesso deste ensaio são os seguintes: 1 Expressão de PRMTs em células de mamíferos, 2 Actividade de PRMTs purificadas, 3 Expressão e purificação de substrato, e 4 completa transferência western das proteínas para a memb...

Metilação proteína foi descrita pela primeira vez em 1968 1. Foi só a primeira clonagem de PRMT1 em 1996, que os pesquisadores começaram a apreciar a importância desta modificação pós-tradução 2. Curiosamente, cerca de 2% de resíduos de arginina em proteínas de extractos nucleares são metilados 3, indicando que a abundância desta modificação. A arginina é um aminoácido carregado positivamente, com uma cadeia lateral básica e os de azoto / s nas cadeias laterais de arginina pode ser modificada após a tradução através da adição de um grupo metilo, um processo conhecido como arginina metilação 4-6. Metilação arginina é catalisada por uma classe de enzimas viz. Transferases de proteína de metilo, arginina (PRMTs). Argininas pode ser ou dimetilado monomethylated e este último pode ser simétrica ou assimétrica, dependendo do tipo de PRMTs que catalisam o processo de 4-6. Proteínas que mostrar argininae-glicina motivos ricos são alvos potenciais para a catálise mediada por PRMT. Metilação mediada por PRMT de substratos foi mostrado para modular a interacção proteína-proteína, interacção proteína-ácido nucleico, a função da proteína, a expressão do gene, e / ou de sinalização celular, as quais são essenciais para a homeostase celular normal 7-9. A fim de entender o papel biológico da proteína arginina metilação, ensaios precisos, eficientes e reproduzíveis são necessários para estabelecer o estado de metilação dos substratos PRMT identificados. No ensaio in vitro de metilação, que avalia a capacidade de catalisar a PRMTs purificados metilação dos seus substratos, é um ensaio bem aceite para o estudo de metilação arginina 10-12. O êxito geral deste ensaio depende em grande parte da actividade dos PRMTs purificadas. PRMTs pode ser expressa e purificada a partir de bactérias ou células de mamífero 10,11. Este ensaio in vitro de metilação, como dPORMENORIZADA na secção de protocolo, baseia-se num método originalmente descrito por Tini e colegas 10. Neste protocolo, que mostram em detalhe os passos envolvidos na expressão e purificação da PRMT1 em células de mamíferos. A capacidade da PRMT1 purificada catalisar a metilação em Ras-activação da GTPase da proteína de ligação da proteína 1 (G3BP1), um substrato conhecido PRMT 8, foi depois avaliado na presença de S-adenosil-L-[metil-3H] metionina como o doador de metilo. Utilizando este ensaio, pode-se definir de forma fiável a capacidade dos PRMTs a novel metilato ou substratos conhecidos, o que é um passo fundamental no estudo da proteína arginina metilação.

<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 metilação é uma das modificações pós-traducionais mais abundantes no núcleo. Proteína arginina metilação pode ser identificada e / ou determinada por meio de métodos de proteómica, e / ou imunotransferência com anticorpos específicos metil-arginina. No entanto, essas técnicas às vezes pode ser enganosa e muitas vezes fornecem resultados falsos positivos. Mais importante, estas técnicas não podem constituir uma prova directa de apoio à especificidade do substrato PRMT. Ensaios in vitro de metilação, por outro lado, são os ensaios bioquímicos, que são sensíveis, e consistentemente se revelar as proteínas identificadas são realmente substratos PRMT. Um ensaio típico de metilação in vitro inclui, PRMTs activa purificada, purificada e substrato marcado com um radioisótopo doador de metilo (S-adenosil-L-[metil-3H] metionina). Aqui, descrevemos um protocolo passo-a-passo para isolar PRMT1 cataliticamente activo, um membro da família PRMT ubiquamente expressa. A mimtilo transferase da PRMT1 purificados foram depois testados em proteína Ras-activação da GTPase de ligação da proteína 1 (G3BP1), um substrato PRMT conhecido, na presença de S-adenosil-L-[metil-3H] metionina como o doador de metilo. Este protocolo pode ser utilizado não só para o estabelecimento do estado de metilação de novos substratos PRMT1 fisiológicos, mas também para a compreensão do mecanismo de base de arginina proteína metilação.

<html> As capacidades de HA-PRMT1 para metilar GST-G3BP1 foram determinadas por um ensaio in vitro de metilação. PRMT1 marcada com HA purificada a partir de lisados ​​de células Beas2B foi utilizado numa reacção de metilação contendo GST-G3BP1, e 1 ^ Ci de S-adenosil-L-[metil-3H] metionina, como um doador de metilo. PRMT1 poderia catalisar eficientemente a metilação de GST-G3BP1 (Figura 1, painel superior). Reacções de metilação, utilizando menus pendentes executadas ...

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 metilação, catalisada por uma classe de enzimas viz., Proteína arginina metil transferases (PRMTs), é o processo de adição enzimática do grupo (s) metilo para argininas dentro proteínas. O ensaio de metilação in vitro é a ferramenta mais confiável para avaliar o estado de metilação de substratos PRMT conhecidos ou novos.

A metilação in vitro Ensaio de Proteína Estudo Arginina metilação

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

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JoVE Journal

Biology

In vitro Methylation Assay to Study Protein Arginine Methylation

15.1K visualizações.  University of Illinois at Chicago.  Proteína arginina metilação, catalisada por uma classe de enzimas viz., Proteína arginina metil transferases (PRMTs), é o processo de adição enzimática do grupo (s) metilo para argininas dentro proteínas. O ensaio de metilação in vitro é a ferramenta mais confiável para avaliar o estado de metilação de substratos PRMT conhecidos ou novos. 

Vídeo: 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.

Teste de sensibilidade visual para a velocidade e direção do movimento em Lagartos

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

Biologia

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.

Determinação de metilação de DNA de genes marcados em Arabidopsis Endosperma

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.

Protein Assay Overlay membrana: um protocolo para testar Interação entre proteínas solúveis e insolúveis 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.

A metilação do DNA: Modificação e Análise de bissulfito

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.

A fase líquida ensaio de captura de afinidade usando pérolas magnéticas para estudar interação proteína-proteína: O Exemplo Poliovírus Nanobody-

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.

A aplicação de um<em&gt; In vitro</em&gt; DNA Assay Proteção ao Visualize Propriedades Mediação stress do 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 ...

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.

Endotelial Tubo celular Formação ensaio para a<em&gt; In Vitro</em&gt; Estudo da angiogênese

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.

Aprimorada Redução Representação Bisulfite Sequencing para a Avaliação de metilação de DNA na resolução de pares de bases

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.