The authors thank Kerstin Glensk for preparing the MTases M.BseCI and Set7/9 and gratefully acknowledge funding by the Excellence Initiative of the German Federal and State Governments and RWTH Aachen University. The authors are happy to provide 6BAz and SeAdoYn or other cofactor analogues for collaborative research.

1. Instruções Gerais <ol><li> Aziridina loja cofactor 6BAz (em DMSO) e proteína MTase SET7 / 9 a -80 ° C e todos os outros reagentes, incluindo co-factor activado SeAdoYn dupla e ADN MTase M.BseCI (em glicerol a 50%) à temperatura de -20 ° C. </li><li> Determinar a concentração de 6BAz e SeAdoYn via UV / Vis utilizando os coeficientes de extinção ε 269nm (6BAz) = 16,000 centímetros -1 M-1 e ε 260nm (SeAdoYn) = 15,400 centímetros -1 M-1 em água deionizada. Determinar a concentração de MTases pelo ensaio de Bradford ou, se o coeficiente de extinção está disponível, por meio de absorção directa a 280 nm. </li><li> Tentar evitar a criação de bolhas por pipetagem intensivo ou em vortex para evitar a perda de actividade da enzima. Em vez disso, misture cuidado pipetando para cima e para baixo. </li><li> Quando a adição de co-factores de aziridina partir de soluções mãe em DMSO a certificar-se de que a concentração final de DMSO no ensaio é de menosdo que 5%. Sempre incluir 10 mM de iões de magnésio no tampão de ensaio para evitar reacções não específicas com o ADN. </li><li> Quando a adição de co-factores activados duplas a partir de soluções de estoque ácidas utilizar pequenos volumes (soluções de reserva altamente concentrada) para evitar a variação do pH e certificar-se de que o pH da solução de ensaio não se altera significativamente. Evitar, por exemplo, tióis, β-mercaptoetanol ou ditiotreitol (DTT), em tampão de ensaio, porque eles podem interferir com a reacção de cliques por complexação dos iões de cobre necessários. </li></ol> 2. Um passo Rotulagem de DNA via Aziridine Cofactores <ol><li> Específica da sequência Induzido por Metiltransferase Etiqueta ing (sorrindo) de DNA plasmídeo com M.BseCI DNA MTase e aziridine cofator 6BAz. <ol><li> Descongelar o cofactor solução a 20 ° C e preparar as misturas de reacção em gelo. </li><li> Além de realizar um ensaio de controlo &quot;cofactor&quot;, para visualizar as modificações não específicas, e um &amp; #8220; enzima &quot;controle, para se certificar de que a preparação MTase está livre do AdoMet cofator natural. </li><li> Para a mistura do ensaio 2 ul de tampão de modificação 10x (contendo 100 mM de Tris-HCl, 100 mM de MgCl 2, 20 mM de β-mercaptoetanol, pH 7,4), 2 uL do pBR322 (0,5 ug / uL), 10 eq. M.BseCI por sequência de reconhecimento no ADN (sequência de reconhecimento em pBR322 1) e o cofactor aziridina 6BAz para uma concentração final de 60 pM num volume total de 20 ul. Adicionar cofator e DNA MTase passada. NOTA: β-mercaptoetanol é tóxico, corrosivo e prejudicial para o ambiente. </li><li> Para o controle &quot;cofator&quot; adicionar água deionizada em vez de M.BseCI e para o controle de &quot;enzima&quot; adicionar água deionizada em vez de 6BAz. </li><li> Misture as soluções cuidado pipetando para cima e para baixo. </li><li> Incubar os tubos a 55 ° C durante 1 h. </li><li> Centrifugar brevemente para recolher todo o líquido na parte inferior dos tubos. </li></ol></li><li> Ensaio restrição-modificação para verificar modificação DNA. <ol><li> Prepara-se uma solução por mistura de 10 ul de tampão 10x R.TaqI (contendo 100 mM de Tris-HCl, 50 mM de MgCl 2, 1 M de NaCl, 1 mg / ml de albumina de soro bovino, pH 8,0), 80 ul de água desionizada e 3,3 mL da endonuclease de restrição (Rease) a partir de Thermus aquaticus (R.TaqI, 10 U / ul). Certifique-se de adicionar o Rease na última etapa. </li><li> Para cada tubo de 2.1.7 adicionar 2 ul de tampão Taq 10x R. e 28 ul da solução a partir de acima (2.2.1). </li><li> Misture as soluções cuidado pipetando para cima e para baixo. </li><li> Incubar os tubos a 65 ° C durante 30 min. </li><li> Centrifugar brevemente para recolher todo o líquido na parte inferior dos tubos. </li></ol></li><li> Teste de desvio de Electromobility (EMSA) com estreptavidina para verificar modificação funcional. <ol><li> Remover 25 mL de cada tubo (2.2.5) e adicionar 2,4 mL de uma solução de estreptavidina (1 mM com respeito a estreptavidina monomer estreptavidina em tampão contendo 100 mM de Na 2 HPO 4, NaCl 100 mM, pH 7,5; 4 equivalentes de biotina total). Adicionar 2,4 mL de tampão de estreptavidina para os restantes tubos. </li><li> Incubar todos os tubos a 37 ° C durante 1 h. </li></ol></li><li> Análise através de electroforese em gel de agarose. <ol><li> Adiciona-se 5 mL de tampão de carregamento 6x (0,25% de azul de bromofenol, 30% de glicerol) a cada tubo. </li><li> Misture delicadamente as soluções. </li><li> Carga de 10 ul de cada amostra para os poços de um gel de agarose (1% de agarose em tampão TBE 0,5x contendo 1x GelRed a partir de uma solução estoque 10.000x). </li><li> Execute o gel em tampão TBE 0,5x com 80 V por aprox. 1 hr. </li><li> Visualizar as bandas de ADN numa mesa de UV (312 nm) com uma câmara CCD equipado com um filtro (540 ± 50 nm). NOTA: a luz UV é prejudicial para os olhos e pele. </li></ol></li></ol> 3. Duas fases de marcação de proteínas via casal Cofactores activados <ol><li> MethyltransfTransferência de Grupos ativado (mTAG) com SET7 / 9 e cofator double-ativado SeAdoYn para histona H3 lisina 4 rotulagem (etapa de modificação) apagar-dirigida. <ol><li> Descongelar os componentes e preparar as misturas de reacção em gelo. NOTA: Sempre mantenha SeAdoYn resfriado para evitar a degradação. </li><li> Além de realizar um ensaio de controlo &quot;cofactor&quot;, para visualizar as modificações não específicas, e um controlo de &quot;enzima&quot;, para excluir as reacções não específicas da sonda fluorescente. </li><li> Prepara-se uma solução de ensaio (20 ul) contendo tampão de modificação (50 mM Tris-HCl, 5% de glicerol, pH 8,5), 10 mM de histona H3, 10 uM SET7 / 9 e 600 uM SeAdoYn (mistura de ambos os epímeros em selénio). Nas últimas etapas adicionar cofator e depois MTase. </li><li> Para o controlo &quot;cofactor&quot; preparar uma solução de ensaio como em 3.1.3 e adicionar AdoMet 60 mM para competir com o cofactor sintético. Para o controle de &quot;enzima&quot; adicionar água deionizada em vez de SeAdoYn.</li><li> Misture as soluções por lentamente pipetagem cima e para baixo. Verificar o pH por adição de 1 ul de cada solução para o campo superior de uma faixa de pH (gama de pH de 5 - 10). </li><li> Incubar a 37 ° C durante 2 horas. </li><li> No gel entretanto preparar um gel de SDS-poliacrilamida a 12% (em execução: 357 mM Bis-Tris pH 6,5-6,8, 0,1% (w / v) de APS, 0,04% (v / v) e TEMED a 12% de acrilamida / bisacrilamida 37,5: 1 ; carregamento do gel: 357 mM Bis-Tris pH 6,5-6,8, 0,1% (w / v) de APS, 0,04% (v / v) e TEMED a 5% de acrilamida / bisacrilamida 37,5: 1). NOTA: A acrilamida / bisacrylamide é tóxico e nocivo à saúde. Usar luvas durante este procedimento. </li></ol></li><li> Rotulagem Chemical de lisina alkinylated 4 em histona H3 via cicloadiï¿½o azida-alcino (CuAAC) (etapa de rotulagem) catalisada por cobre. <ol><li> Pouco antes do fim da reacção de modificação preparar uma mistura 5x clique contendo 3 mM de CuSO 4, 3 mM de tris (3-hidroxipropil-triazolilmetil) amina (THPTA), ascorbato de sódio 250 mM e TAMRA 6 mM de azida com umvolume total de 20 ul. </li><li> Adiciona-se 5 uL da mistura de 5x clique recentemente preparada a cada tubo para iniciar a CuAAC e extingue-se a reacção de modificação. </li><li> Misture delicadamente por pipetagem cima e para baixo. </li><li> Proteja todos os tubos com folha de alumínio da luz para evitar a foto-branqueamento do fluoróforo. </li><li> Incubar a 20 ° C durante 1 h. </li></ol></li><li> Precipitação de proteínas para remover o excesso de livre fluorophore TAMRA. <ol><li> Para evitar resplendor da marcação fluorescente histona H3 por intensivo fluorescência in-gel de livre fluorophore TAMRA, retire o excesso fluorophore por precipitação de proteínas (3.3.2 - 3.3.4) 34. </li><li> Adicionar 75 mL de metanol, 18,8 mL de clorofórmio e 50 ul de água desionizada a cada tubo e vortex brevemente após cada adição. Centrifuga-se a 16000 xg durante 5 min. Remover a fase superior sem perturbar a camada de interface, que contém a proteína. </li><li> Adicionar 56,3 mL de metanol à fase restante in cada tubo, vortex e centrifuga-se a 16.000 xg durante 5 min para sedimentar a proteína. Remover o sobrenadante. Repetir esta etapa para lavar o sedimento. </li><li> Cubra os tubos abertos com um tecido que não solte fiapos e deixe-os secar por 15-30 min. </li></ol></li><li> Análise via SDS PAGE. <ol><li> Dissolve-se as proteínas precipitadas a partir de 3.3.4 em 20 ul de tampão de carga SDS (50 mM Tris-HCl, 2,5% (w / v) de SDS, 10% (v / v) de glicerol, 320 mM β-mercaptoetanol e 0,05% (w / v) azul de bromofenol, pH 6,8). Certifique-se para dissolver completamente o sedimento, por lavagem das paredes dos tubos com uma pipeta. </li><li> Incubar as amostras a 95 ° C durante 10 min e deixa-os arrefecer até 20 ° C. </li><li> Centrifugar brevemente para recolher todo o líquido na parte inferior dos tubos. </li><li> Coloque toda a quantidade de cada amostra para os poços de um gel de poliacrilamida SDS (3.1.7). Use 50 mM de MOPS, 50 mM de Tris-X (Tris-base), EDTA 5 mM, 0,1% (w / v) de SDS como tampão de corrida para electroforese. </li><li>Execute o gel com 120 V por aprox. 90 min. </li><li> Visualizar a fluorescência em gel sobre uma mesa de UV (312 nm) com uma câmara CCD equipado com um filtro (540 nm ± 50 nm). NOTA: a luz UV é prejudicial para os olhos e pele. </li></ol></li></ol>

The authors disclose the following competing financial interest: E.W. is inventor on related patents.

Rotulagem de uma etapa de DNA com MTases DNA e cofatores aziridina (DNA sorrindo) é um método robusto, mas alguns aspectos devem ser considerados quando se planeja o experimento. Cofactor aziridina: A concentração 6BAz para marcação de ADN com M.BseCI foi de 60 uM. Ao usar outros MTases DNA a concentração cofator deve ser otimizada, por exemplo, concentrações tão baixas como 20? M têm sido empregadas com o DNA MTase M.TaqI 19. Baixas concentrações 6...

Rotulagem específica de ácidos nucléicos e proteínas 1,2 3,4 é de grande interesse para caracterizações funcionais, diagnóstico médico e (nano) biotecnologia. Aqui apresentamos um método de marcação enzimática para esses biopolímeros que é baseada no S -adenosyl-l-metionina (AdoMet ou SAM) methyltransferases -dependentes (MTases). Esta classe de enzimas (EC 2.1.1.) Tem como alvo as posições individuais nucleofílicos (azoto, oxigénio, enxofre e átomos de carbono) no interior resíduos específicos de ácidos nucleicos e proteínas e naturalmente transfere o grupo metilo activado da AdoMet cofactor (Figura 1A) 5. Além disso, pode utilizar MTases análogos sintéticos de cofactor para marcação específica com fluoróforos, marcadores de afinidade ou outras etiquetas (Figura 1B) 6. Duas classes de análogos da AdoMet foram desenvolvidos: cofactores aziridina para M ethyltransferase- específico equence S-I </strong&gt; Nduced L abel ING (sorrindo) 7 e duplos análogos ativados AdoMet para dirigida por ethyltransferase m T ransferência de A ctivated rupos G (mTAG) 8. <img alt="A Figura 1" fo:content-width="7in" src="/files/ftp_upload/52014/52014fig1highres.jpg" width="700" /> Figura 1: As reacções catalisadas por metiltransferases (MTases) de transferência de grupo A. Metil do cofactor naturais AdoMet (SAM) a vários substratos, incluindo ADN, ARN, proteínas e pequenas biomoléculas B. Etiquetagem / funcionalização de ácidos nucleicos e proteínas (NNNNN. =. pares de bases de DNA, nucleotídeos de RNA para ácidos e aminoácidos das proteínas; XXXXX = sequência de reconhecimento do MTase com resíduo alvo em verde) com análogos cofactor sintéticos. Cofatores Aziridine contendo um grupo repórter (esfera azul)ligado ao anel de adenina são especificamente sequência acoplado com o resíduo alvo (esquerda) e análogos dupla activada AdoMet conduzir à transferência de cadeias alquilo que transportam um repórter estendidos química Y (direita), que podem ser marcados por reacção bioorthogonal clique num segundo passo. <a href="https://www.jove.com/files/ftp_upload/52014/52014fig1highres.jpg" target="_blank">Por favor, clique aqui para ver uma versão maior desta figura.</a> Cofatores Aziridine trabalhar melhor com DNA MTases. Eles contêm um anel de três membros, com um átomo de azoto, 9 (ou um seu N mostarda 10,11) em vez do centro do grupo de sulfónio como reactivo. A protonação do átomo de azoto presente no anel de aziridina activa para o ataque nucleofílico pelo nucleotídica alvo que leva para o acoplamento covalente de todo o co-factor com ADN. Ao ligar-se grupos repórter para o anel de adenina os cofactores de aziridina pode ser utilizado em combinação com DNA para rotular MTases ADN em um passo ( <stron<html> g&gt; Figura 1B, à esquerda) 7,12. Isto é demonstrado em detalhe para a biotinilação de ADN com 6BAz 13-15 (cofactor aziridina com biotina ligada à posição 6 do anel de adenina) e a adenina-specific DNA MTase de Bacillus stearothermophilus (M.BseCI) 16 (Figura 2, consulte a seção de protocolo 2: rotulagem de uma etapa de DNA via aziridina cofatores). Além M.BseCI (&#39;sequência de reconhecimento, o MTases ADN de Thermus aquaticus (M.TaqI, 5&#39;-TCG Um -3 5&#39;-ATCG A T-3)&#39;), a partir de Haemophilus heamolyticus (M.HhaI, 5 &#39;-G C GC-3&#39;) e de Spiroplasma (M.SssI, 5&#39;-G C-3 &#39;) foram usados ​​com sucesso para biotinilar ADN com 6BAz 17. Além disso, co-factores de aziridina pode ser empregue para um passo de ADN de fluorescência rotulagem 18,19. ontent &quot;fo: manter-together.within-page =&quot; always &quot;&gt; <img alt="A Figura 2" fo:content-width="7in" src="/files/ftp_upload/52014/52014fig2highres.jpg" width="700" /> Figura 2:. Sequência de biotinilação específica de um passo de ADN com M.BseCI e 6BAz O ADN MTase M.BseCI reconhece a sequência de ADN de cadeia dupla 5&#39;-ATCG A T-3 &#39;e naturalmente metila o grupo amino da segunda adenina resíduo (verde) usando AdoMet. Com o co-fator aziridine 6BAz o curso da reacção é alterada e M.BseCI leva para sequenciar biotinilaï¿½o DNA específico acoplando todo o cofator incluindo biotina (azul) com a adenina alvo. <a href="https://www.jove.com/files/ftp_upload/52014/52014fig2highres.jpg" target="_blank">Por favor, clique aqui para ver uma versão maior desta figura.</a> Duplos análogos AdoMet activados contêm estendido cadeias laterais insaturadas em vez de um grupo metilo no centro de sulfónio (Figura 1B </strong&gt;, direita) 20. A ligação dupla ou tripla insaturada em β-position para o centro da sulfonium compensa eletronicamente efeitos estéricos desfavoráveis ​​no interior do estado de transição de estabilização conjugative. Uma vez que tanto o centro de sulfonium ea ligação insaturada ativar a cadeia lateral para a transferência enzimática, estes cofatores foram nomeados análogos AdoMet double-ativadas. Tipicamente, eles são utilizados para transferir as cadeias laterais com grupos repórteres únicos químicos (químicos), como grupos amino, alquino e azida, para rotulagem quimio-selectiva numa segunda etapa 8,21. Em geral, os análogos dupla ativada AdoMet pode não só funcionam como co-fatores para DNA MTases 8,20,21, mas também para RNA MTases 22,23 e MTases proteína 24-28 permitindo rotulagem adicional de RNA e proteínas. No entanto, as cadeias laterais estendidas estão estericamente mais exigente do que um grupo metilo e alargando os locais activos MTAse por engenharia de proteínas é frequentementeen necessária para a obtenção de taxas de transferência eficientes. Outra solução para este problema é a utilização de um análogo AdoMet com um grupo pequeno propargilico (três carbonos), onde o terminal de alcino tem duas funções: 1. Estabilização do estado de transição durante a transferência enzimática e 2. alça reativos para seguir modificações químicas por cobre- catalisada cicloadiï¿½o azida-alcino (CuAAC) clique química. Descobriu-se que o resultante propargílica AdoMet análogo 29 é bastante instável em condições neutras ou ligeiramente básicas e somente de uso limitado. Este inconveniente pode ser corrigido, substituindo o átomo de enxofre com selênio. O cof actor resultante 5 &#39;- [(Se) [(3 S) -3-amino-3-carboxipropil] prop-2-ynylselenonio] -5&#39;-desoxiadenosina (SeAdoYn, Figura 3) é aceite pelo ADN de tipo selvagem, ARN e MTases proteína 30-32 que anula a necessidade de engenharia de proteínas, em muitos casos. Isto é exemplificado por fluorescência pró<html> rotulagem tein com a lisina histona H3 4 (H3K4) MTase SET7 / 9 33 (Figura 3, ver seção de protocolo 3: marcação de proteínas em dois passos via cofatores ativados duplas). <img alt="Figura 3" fo:content-width="7in" src="/files/ftp_upload/52014/52014fig3highres.jpg" width="700" /> Figura 3:. Específica da sequência de fluorescência de duas etapas rotulagem de histona H3 com SET7 / 9, SeAdoYn e TAMRA azida A proteína MTase SET7 / 9 naturalmente metila o grupo amino da lisina 4 em histona H3 (H3K4, verde) usando AdoMet. Com o co-fator duplo ativado SeAdoYn o MTase transfere um grupo propargilico pequeno (vermelho) ao resíduo de lisina. A ligação tripla terminal conectado é então modificada seletivamente em uma reação clique bioorthogonal (azida-alcino cicloadiï¿½o catalisada por cobre, CuAAC) com (tetramethylrhodamine, azul) fluorophore derivado-azida TAMRA.carga / 52014 / &quot;target =&quot; _ 52014fig3highres.jpg blank &quot;&gt; Clique aqui para ver uma versão maior desta figura.

<table border="0" cellpadding="0" cellspacing="0" width="669">
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			<td height="21" style="height:21px;width:245px;">Name of Reagent/ Equipment</td>
			<td style="width:139px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">6BAz</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>Synthesized according to Weinhold et al., Patent number US 8,129,106, published March 6, 2012.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">&#946;-Mercaptoethanol</td>
			<td style="width:139px;">Serva</td>
			<td align="right" style="width:119px;">28625</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Acetic acid</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td align="right" style="width:119px;">10304980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Acrylamide/Bis Solution, 37.5:1</td>
			<td style="width:139px;">Serva</td>
			<td align="right" style="width:119px;">10688</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">UltraPure Agarose</td>
			<td style="width:139px;">Invitrogen</td>
			<td align="right" style="width:119px;">16500100</td>
			<td></td>
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			<td height="21" style="height:21px;width:245px;">Ammonium persulfate (APS)</td>
			<td style="width:139px;">Serva</td>
			<td align="right" style="width:119px;">13375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Bis-Tris</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1304</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Boric acid</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Bromophenol blue Na salt</td>
			<td style="width:139px;">Serva</td>
			<td align="right">15375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Copper(II) sulfate</td>
			<td style="width:139px;">Aldrich</td>
			<td style="width:119px;">C1297</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Chloroform</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td align="right">10020090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Coomassie Brilliant Blue</td>
			<td style="width:139px;">Serva</td>
			<td align="right" style="width:119px;">17525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">EDTA disodium salt</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Ethanol</td>
			<td style="width:139px;">Merck</td>
			<td align="right" style="width:119px;">100983</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">GelRed (10000x in water)</td>
			<td style="width:139px;">Biotium</td>
			<td align="right" style="width:119px;">41003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Glycerol (99.5%)</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">2006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">FastRuler Low Range DNA Ladder</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td style="width:119px;">SM1103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Histone H3</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>Expressionplasmid obtained from Dr. Philipp Voigt and Prof. Danny Reinberg; expression and isolation according to T. J. Richmond et al., J. Mol. Biol. 1997, 272, 301-311.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">M.BseCI</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>Expressionplasmid obtained from Dr. Michael Kokkinidis; expression and isolation according to Kapetaniou et al., Acta Cryst. 2006, F63, 12-14.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Methanol</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td align="right" style="width:119px;">10675112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Magnesiumchloride&#160; hexahydrate</td>
			<td style="width:139px;">J.T. Baker</td>
			<td align="right" style="width:119px;">4003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">MOPS</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Sodium chloride</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">pH strip (Neutralit)</td>
			<td style="width:139px;">Merck</td>
			<td align="right" style="width:119px;">1,095,330,001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">pBR322</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td style="width:119px;">SD0041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">R.TaqI (10u/&#181;l)</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td>ER0671</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">SeAdoYn</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>Synthesized according to Willnow et al., ChemBioChem 2012, 13, 1167-1173.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Set7/9</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>Expressionplasmid obtained from Prof. Danny Reinberg, expression and isolation according to D. Reinberg et al., Genes Dev.2002, 16, 479-489.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Streptavidin</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">3058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">(+)-Sodium L-ascorbate</td>
			<td style="width:139px;">Sigma Life Science</td>
			<td style="width:119px;">A7631</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">SDS Granular</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1833</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">di-Sodiumhydrogenphosphat</td>
			<td style="width:139px;">Merck</td>
			<td align="right" style="width:119px;">106,586</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">TAMRA-azide</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">TaqI buffer (10x)</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td style="width:119px;">B28</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:245px;">N,N,N&#39;,N&#39;-Tetramethylethylenediamine (TEMED)</td>
			<td style="width:139px;">Acros Organics</td>
			<td align="right" style="width:119px;">42058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Tris-HCl</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Tris-X (TRIS-base)</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1018</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">Tris(3-hydroxypropyltriazolyl-methyl)amine (THPTA)</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">762342</td>
			<td></td>
		</tr>
	</tbody>
</table>

sequence-specific labeling of nucleic acids and proteins with methyltransferases and cofactor analogues

-Adenosyl S-L-metionina (AdoMet ou SAM) metiltransferases dependentes de (MTAse) catalisam a transferência do grupo metilo de AdoMet activado a posições específicas no ADN, ARN, proteínas e pequenas biomoléculas. Esta reacção de metilação pode ser natural, expandida para uma grande variedade de reacções de alquilação usando análogos sintéticos de cofactor. A substituição do centro reactivo da AdoMet sulfónio com um anel aziridina leva à co-factores que podem ser acoplados com ADN por vários MTases ADN. Estes cofactores de aziridina pode ser equipado com grupos repórter em diferentes posições a porção de adenina e usado para S-specific equence M ethyltransferase- I nduced ing L abel de DNA (DNA de sorriso). Como um exemplo típico, damos um protocolo para a biotinilação de DNA de plasmídeo pBR322 na sequência 5&#39;-ATCG A T-3 &#39;com o ADN MTase M.BseCI e o cofactor aziridina em 6BAzum passo. Extensão do grupo metil ativado com insaturados grupos alquilo resulta em uma outra classe de análogos AdoMet que são utilizados para dirigida por ethyltransferase m T ransferência de A ctivated rupos G (mTAG). Uma vez que as cadeias laterais estendidas são activados pelo centro de sulfónio e a ligação insaturada, estes cofactores são chamados análogos AdoMet dupla activada. Estes análogos não só funcionam como co-factores para MTases ADN, como os cofactores de aziridina, mas também para ARN, proteínas e pequenas MTases molécula. Eles são tipicamente usados ​​para a modificação enzimática da MTAse substratos com grupos funcionais únicos que são marcados com grupos repórter num segundo passo químico. Isso é exemplificado em um protocolo para a rotulagem de fluorescência da proteína histona H3. Um pequeno grupo propargilo é transferido do SeAdoYn cofactor análogo à proteína pela lisina histona H3 4 (H3K4) MTase SET7 / 9 seguido por clique rotulagem dohistona H3 alkynylated com TAMRA azida. Rotulagem MTase mediada com análogos cofactor é uma tecnologia capacitadora para muitas aplicações interessantes, incluindo a identificação e estudo funcional de substratos MTase bem como genotipagem de DNA e detecção de metilação.

Um passo-a Rotulagem de DNA via Aziridine Cofactores Esta reacção é levada a cabo exemplo com o ADN MTase M.BseCI, que modifica o resíduo de adenina segunda dentro do 5&#39;-ATCG A T-3 &#39;sequência de cadeia dupla e tem um local de reconhecimento no plasmídeo pBR322 (Figura 4A). Para testar rotulagem plasmídeo, pBR322 é desafiado com a endonuclease de restrição (Rease) R.TaqI (5&#39;-TCGA-3 &#39;). R.TaqI tem sete sites na pBR322, um dos...

Watch this Scientific Journal Video about Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues at JoVE.com

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

DNA e as proteínas são seqüência especificamente marcado com afinidade ou grupos repórter fluorescentes de DNA ou proteína methyltransferases e análogos sintéticos cofactor. Dependendo da especificidade do cofactor de enzimas, aziridina ou duplas análogos de cofactores activados são empregues para marcação de uma ou duas etapas.

Rotulagem específica de seqüência de ácidos nucléicos e proteínas com Metiltransferases e cofator Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

sequence-specific-labeling-nucleic-acids-proteins-with

Research

JoVE Journal

Biology

13.9K visualizações.  RWTH Aachen University.  DNA e as proteínas são seqüência especificamente marcado com afinidade ou grupos repórter fluorescentes de DNA ou proteína methyltransferases e análogos sintéticos cofactor. Dependendo da especificidade do cofactor de enzimas, aziridina ou duplas análogos de cofactores activados são empregues para marcação de uma ou duas etapas. 

Vídeo: Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

Immunofluorescent Detection of Two Thymidine Analogues (CldU and IdU) in Primary Tissue

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are analyzed. Established methods to detect cell division include direct visualization by continuous microscopy in cell culture, dilution of vital dyes such as carboxy&#64258;uorescein di-aetate succinimidyl ester (CFSE), immuno-detection of mitogenic antigens such as ki67 or PCNA, and thymidine analogues. Thymidine analogues can be detected by a variety of methods including radio-detection for tritiated thymidine, immuno-detection for bromo-deoxyuridine (BrdU), chloro-deoxyuridine (CldU) and iodo-deoxyuridine (IdU), and chemical detection for ethinyl-deoxyuridine (EdU). We have derived a strategy to detect sequential incorporation of different thymidine analogues (CldU and IdU) into tissues of adult mice. Our method allows investigators to accurately quantify two successive rounds of cell division. By optimizing immunostaining protocols our approach can detect very low dose thymidine analogues administered via the drinking water, safe to administer to mice for prolonged periods of time. Consequently, our technique can be used to detect cell turnover in very long-lived tissues. Optimal immunofluoresent staining results can be achieved in multiple tissue types, including pancreas, skin, gut, liver, adrenal, testis, ovary, thyroid, lymph node, and brain. We have also applied this technique to identify oncogenic transformation within tissues. We have further applied this technique to determine if transit-amplifying cells contribute to growth or renewal of tissues. In this sense, sequential administration of thymidine analogues represents a novel approach for studying the origins and survival of cells involved in tissue homeostasis.

Immunofluorescent Detection of Two Thymidine Analogues CldU and IdU in Primary Tissue

We have derived a strategy to detect sequential incorporation of thymidine analogues (CldU and IdU) into tissues of adult mice to quantify two successive rounds of cell division. This strategy is useful to detect cell turnover of long-lived tissues, oncogenic transformation, or transit-amplifying cells.

Detecção de imunofluorescência de dois análogos da timidina (CldU e UDI), em tecido primário

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are ...

Biologia

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

Perfil de metiltransferases e Outros S-Adenosil- L Homocisteína-Proteínas de Ligação de captura por Espectrometria de Massa Compound (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

NanoDrop quantificação Microvolume de Ácidos Nucléicos

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

Examinando o Dynamics Conformacional de Proteínas de Membrana In situ Com Labeling-Site dirigido Fluorescência

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

Rotulagem de linhagem de células Zebrafish com Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF &middot; (OD260-OD320)&middot; NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1.
Protein concentration results can be expressed as &mu;g/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.

This communication presents data on the accuracy and reproducibility of the BioSpec-nano UV-VIS spectrophotometer for dsDNA and protein quantitation. Even with ultra-small volumes (1 to 2 L), reproducibility is excellent, while the automated wiping function improves throughput and results in minimal carryover for more precise results.

Determinação de concentração de ácidos nucléicos e proteínas Usando o Micro-volume Bio-spec Espectrofotômetro Nano

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells. While fluorescent 
proteins such as green fluorescence protein (GFP) have been extensively used as fusion partners to proteins to track the properties of a protein of interest1, recent 
developments with smaller tags enable new functionalities of proteins to be examined in cells such as conformational change and protein-association 2, 3. One small 
tag system involves a tetracysteine motif (CCXXCC) genetically inserted into a target protein, which binds to biarsenical dyes, ReAsH (red fluorescent) and FlAsH 
(green fluorescent), with high specificity even in live cells 2. The TC/biarsenical dye system offers far less steric constraints to the host protein than 
fluorescent proteins which has enabled several new approaches to measure conformational change and protein-protein interactions 4-7. We recently developed 
a novel application of TC tags as sensors of oligomerization in cells expressing mutant huntingtin, which when mutated aggregates in neurons in Huntington 
disease 7. Huntingtin was tagged with two fluorescent dyes, one a fluorescent protein to track protein location, and the second a TC tag which only 
binds biarsenical dyes in monomers. Hence, changes in colocalization between protein and biarsenical dye reactivity enabled submicroscopic oligomer 
content to be spatially mapped within cells. Here, we describe how to label TC-tagged proteins fused to a fluorescent protein (Cherry, GFP or CFP) 
with FlAsH or ReAsH in live mammalian cells and how to quantify the two color fluorescence (Cherry/FlAsH, CFP/FlAsH or GFP/ReAsH combinations).

The biarsenical dyes FlAsH and ReAsH bind specifically to tetracysteine motifs in proteins and can selectively label proteins in live cells. Recently this labeling strategy has been used to develop sensors for different protein conformations or oligomeric states. We describe the labeling approach and methods to quantitatively analyze binding.

ReAsH / Flash Rotulagem e Análise de Imagem Sensor de Proteínas em Células Tetracysteine

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells.  While fluorescent 
proteins ...

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine cell central metabolism and discover new enzymes is 13C-assisted metabolism analysis 1. This technique is based on isotopic labeling, whereby microbes are fed with a 13C labeled substrates. By tracing the atom transition paths between metabolites in the biochemical network, we can determine functional pathways and discover new enzymes. 
As a complementary method to transcriptomics and proteomics, approaches for isotopomer-assisted analysis of metabolic pathways contain three major steps 2. First, we grow cells with 13C labeled substrates. In this step, the composition of the medium and the selection of labeled substrates are two key factors. To avoid measurement noises from non-labeled carbon in nutrient supplements, a minimal medium with a sole carbon source is required. Further, the choice of a labeled substrate is based on how effectively it will elucidate the pathway being analyzed. Because novel enzymes often involve different reaction stereochemistry or intermediate products, in general, singly labeled carbon substrates are more informative for detection of novel pathways than uniformly labeled ones for detection of novel pathways3, 4. Second, we analyze amino acid labeling patterns using GC-MS. Amino acids are abundant in protein and thus can be obtained from biomass hydrolysis. Amino acids can be derivatized by N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) before GC separation. TBDMS derivatized amino acids can be fragmented by MS and result in different arrays of fragments. Based on the mass to charge (m/z) ratio of fragmented and unfragmented amino acids, we can deduce the possible labeled patterns of the central metabolites that are precursors of the amino acids. Third, we trace 13C carbon transitions in the proposed pathways and, based on the isotopomer data, confirm whether these pathways are active 2. Measurement of amino acids provides isotopic labeling information about eight crucial precursor metabolites in the central metabolism. These metabolic key nodes can reflect the functions of associated central pathways.
13C-assisted metabolism analysis via proteinogenic amino acids can be widely used for functional characterization of poorly-characterized microbial metabolism1. In this protocol, we will use Cyanothece 51142 as the model strain to demonstrate the use of labeled carbon substrates for discovering new enzymatic functions.

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

13C-isotope labeling is a useful technique for determining the cell central metabolism for various types of microorganisms. After cells have been cultured with a specific labeled substrate, GC-MS measurement can reveal functional metabolic pathways based on unique labeling patterns in proteinogenic amino acids.

Confirmação via metabólica e descoberta através 13 C-rotulagem de proteinogénicos Aminoácidos

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

Medindo a progressão do ciclo celular Cinética com marcação metabólica e Citometria de Fluxo

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

Analyzing and Building Nucleic Acid Structures with 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures. Protocol 1 lists the set of instructions needed to download and install the software. This is followed, in Protocol 2, by the analysis of a nucleic acid structure, including the assignment of base pairs and the determination of rigid-body parameters that describe the structure and, in Protocol 3, by a description of the reconstruction of an atomic model of a structure from its rigid-body parameters. The most recent version of 3DNA, version 2.1, has new features for the analysis and manipulation of ensembles of structures, such as those deduced from nuclear magnetic resonance (NMR) measurements and molecular dynamic (MD) simulations; these features are presented in Protocols 4 and 5. In addition to the 3DNA stand-alone software package, the w3DNA web server, located at <a href="http://w3dna.rutgers.edu" target="_blank">http://w3dna.rutgers.edu</a>, provides a user-friendly interface to selected features of the software. Protocol 6 demonstrates a novel feature of the site for building models of long DNA molecules decorated with bound proteins at user-specified locations.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures.

Análise e construção de estruturas de Ácido Nucleico com 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...