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. Instrucciones generales <ol><li> Aziridínico tienda cofactor 6BAz (en DMSO) y la proteína MTase Set7 / 9 a -80 ° C y todos los demás reactivos incluidos cofactor doble activado SeAdoYn y ADN MTase M.BseCI (en el 50% de glicerol) a -20 ° C. </li><li> Determinar la concentración de 6BAz y SeAdoYn mediante espectroscopia UV / Vis utilizando los coeficientes de extinción ε 269 nm (6BAz) = 16,000 cm -1 M -1 y ε 260 nm (SeAdoYn) = 15,400 cm -1 M -1 en agua desionizada. Determinar la concentración de MTases por el ensayo de Bradford o, si el coeficiente de extinción está disponible, a través de la absorción directa a 280 nm. </li><li> Trate de evitar la creación de burbujas pipeteando intensiva o vórtex para evitar la pérdida de la actividad enzimática. En su lugar, se mezcla con la pipeta suavemente hacia arriba y hacia abajo. </li><li> Cuando la adición de cofactores de aziridina partir de soluciones madre en DMSO asegurarse de que la concentración final de DMSO en el ensayo es menosal 5%. Siempre incluya 10 iones de magnesio mM en el tampón de ensayo para evitar reacciones inespecíficas con ADN. </li><li> Cuando la adición de cofactores dobles activados partir de soluciones madre ácidas usar pequeños volúmenes (soluciones madre altamente concentradas) para evitar cambios de pH y asegúrese de que el pH de la solución de ensayo no cambia significativamente. Evitar tioles, por ejemplo, β-mercaptoetanol o ditiotreitol (DTT), en el tampón de ensayo, ya que pueden interferir con la reacción de clic por complejación de los iones de cobre requeridas. </li></ol> 2. Un paso Etiquetado de ADN a través de aziridina cofactores <ol><li> Etiqueta ing Metiltransferasa inducido-secuencia específica (sonriendo) de ADN plásmido con M.BseCI ADN MTase y cofactor aziridínico 6BAz. <ol><li> Descongelar la solución de cofactor a 20 ° C y preparar las mezclas de reacción en hielo. </li><li> Además del ensayo de realizar un control &quot;cofactor&quot;, para visualizar las modificaciones no específicas, y un &amp; #8220; enzima &quot;control, para asegurarse de que la preparación MTase está libre de la AdoMet cofactor natural. </li><li> Para la mezcla de ensayo de 2 l de tampón 10X modificación (que contiene 100 mM Tris-HCl, 100 mM MgCl2, 20 mM β-mercaptoetanol, pH 7,4), 2 l de pBR322 (0,5 g / l), 10 eq. M.BseCI por secuencia de reconocimiento en el ADN (secuencia 1 reconocimiento en pBR322) y el cofactor aziridina 6BAz a una concentración final de 60 micras dentro de un volumen total de 20 l. Añadir cofactor y el ADN MTase pasado. NOTA: β-mercaptoetanol es tóxico, corrosivo y perjudicial para el medio ambiente. </li><li> Para el control &quot;cofactor&quot; añadir agua desionizada en lugar de M.BseCI y para el control &quot;enzima&quot; añadir agua desionizada en lugar de 6BAz. </li><li> Mezclar las soluciones pipeteando suavemente hacia arriba y hacia abajo. </li><li> Incubar los tubos a 55 ° C durante 1 hr. </li><li> Centrifugar brevemente para recoger todo el líquido en la parte inferior de los tubos. </li></ol></li><li> Ensayo de restricción-modificación para verificar la modificación del ADN. <ol><li> Preparar una solución mediante la mezcla de 10 l de 10x tampón R.TaqI (que contiene 100 mM de Tris-HCl, 50 mM de MgCl 2, 1 M NaCl, 1 mg / ml de albúmina de suero bovino, pH 8,0), 80 l de agua desionizada y 3,3 l de la endonucleasa de restricción (REase) a partir de Thermus aquaticus (R.TaqI, 10 U / l). Asegúrese de añadir el REase en el último paso. </li><li> A cada tubo de 2.1.7 añadir 2 l de tampón 10X Taq I y R. 28 l de la solución desde arriba (2.2.1). </li><li> Mezclar las soluciones pipeteando suavemente hacia arriba y hacia abajo. </li><li> Incubar los tubos a 65 ° C durante 30 min. </li><li> Centrifugar brevemente para recoger todo el líquido en la parte inferior de los tubos. </li></ol></li><li> Ensayo de cambio de movilidad eléctrica (EMSA) con estreptavidina para verificar modificación funcional. <ol><li> Retire 25 l de cada tubo (2.2.5) y añadir 2,4 l de una solución de estreptavidina (1 mM con respecto a la estreptavidina monomer estreptavidina en tampón que contiene 100 mM Na 2 HPO 4, NaCl 100 mM, pH 7,5; 4 equivalentes de biotina total). Añadir 2,4 l de tampón de estreptavidina a los tubos restantes. </li><li> Incubar todos los tubos a 37 ° C durante 1 hr. </li></ol></li><li> Análisis mediante electroforesis en gel de agarosa. <ol><li> Añadir 5 l de tampón de carga 6x (0,25% azul de bromofenol, 30% de glicerol) a cada tubo. </li><li> Mezclar las soluciones suavemente. </li><li> Cargar 10 l de cada muestra en los pocillos de un gel de agarosa (1% de agarosa en tampón TBE 0,5x contiene 1x GelRed de una solución 10.000x de valores). </li><li> Ejecute el gel en tampón TBE 0,5x con 80 V durante aprox. 1 hr. </li><li> Visualizar bandas de ADN en un vector de UV (312 nm) con una cámara CCD equipada con un filtro (540 ± 50 nm). NOTA: La luz UV es perjudicial para los ojos y la piel. </li></ol></li></ol> 3. Dos etapas de etiquetado de proteínas a través de cofactores dobles Activado <ol><li> MethyltransfTransferencia de grupos activados (MTag) con Set7 / 9 y cofactor de doble activado SeAdoYn para la histona H3 lisina 4 etiquetado (etapa de modificación) borrar-Dirigida. <ol><li> Descongele los componentes y preparar las mezclas de reacción en hielo. NOTA: Mantenga siempre SeAdoYn enfría para evitar la degradación. </li><li> Además del ensayo de realizar un control &quot;cofactor&quot;, para visualizar las modificaciones no específicas, y un control &quot;enzima&quot;, para excluir reacciones no específicas de la sonda fluorescente. </li><li> Preparar una solución de ensayo (20 l) que contenía tampón de modificación (mM Tris-HCl 50, 5% de glicerol, pH 8,5), 10 mM de histona H3, 10 mM Set7 / 9 y 600 mM SeAdoYn (mezcla de ambos epímeros en selenio). En los últimos pasos añadir cofactor y luego MTase. </li><li> Para el control &quot;cofactor&quot; preparar una solución de ensayo como en 3.1.3 y añadir 60 mM AdoMet para competir con el cofactor sintético. Para el control &quot;enzima&quot; añadir agua desionizada en lugar de SeAdoYn.</li><li> Mezclar las soluciones con la pipeta lentamente hacia arriba y hacia abajo. Comprobar el pH mediante la adición de 1 l de cada solución para el campo superior de una tira de pH (intervalo de pH de 5 - 10). </li><li> Incubar a 37 ° C durante 2 hr. </li><li> En el gel Mientras tanto preparar un gel de poliacrilamida SDS 12% (que se ejecuta: 357 Bis-Tris pH 6.5 a 6.8 mM, 0,1% (w / v) APS, 0,04% (v / v) TEMED y 12% de acrilamida / bisacrilamida 37,5: 1 ; gel de carga: 357 Bis-Tris pH 6.5 a 6.8 mM, 0,1% (w / v) APS, 0,04% (v / v) TEMED y 5% de acrilamida / bisacrilamida 37,5: 1). NOTA: La acrilamida / bisacrilamida es tóxico y la salud peligrosos. Use guantes durante este procedimiento. </li></ol></li><li> Etiquetado química de lisina alkinylated 4 en la histona H3 mediante cicloadición-azida alquino (CuAAC) (etapa de marcaje) catalizada por cobre. <ol><li> Justo antes del final de la reacción de modificación preparar una mezcla 5x clic que contiene 3 mM CuSO 4, 3 mM tris (3-hidroxipropil-triazolilmetilo) amina (THPTA), ascorbato de sodio 250 mM y 6 mM TAMRA azida con unavolumen total de 20 l. </li><li> Añadir 5 l de la mezcla 5x clic recién preparado a cada tubo para iniciar la CuAAC y apagar la reacción de modificación. </li><li> Mezclar suavemente con la pipeta hacia arriba y hacia abajo. </li><li> Proteger a todos los tubos con papel de aluminio de la luz para evitar la foto-blanqueo del fluoróforo. </li><li> Incubar a 20 ° C durante 1 hr. </li></ol></li><li> Precipitación de proteínas para eliminar el exceso de libre fluoróforo TAMRA. <ol><li> Para evitar resplandor de la etiqueta fluorescente histona H3 por intensa fluorescencia en gel de libre fluoróforo TAMRA, eliminar el exceso de fluoróforo por precipitación de proteínas (3.3.2 - 3.3.4) 34. </li><li> Añadir 75 l de metanol, 18,8 l de cloroformo y 50 l de agua desionizada a cada tubo y vórtice brevemente después de cada adición. Centrifugar a 16.000 xg durante 5 min. Eliminar la fase superior sin molestar a la capa de interfaz, que contiene la proteína. </li><li> Añadir 56,3 l de metanol a la fase restante in cada tubo, vórtice y se centrifuga a 16.000 xg durante 5 min para sedimentar la proteína. Eliminar el sobrenadante. Repita este paso para lavar el pellet. </li><li> Cubra los tubos abiertos con un pañuelo de papel libre de pelusa y deje que se sequen durante 15 - 30 min. </li></ol></li><li> Análisis mediante SDS PAGE. <ol><li> Disolver las proteínas precipitadas a partir de 3.3.4 en 20 l de tampón SDS de carga (50 mM Tris-HCl, 2,5% (w / v) SDS, 10% (v / v) de glicerol, 320 mM β-mercaptoetanol y 0,05% (w / v) de azul de bromofenol, pH 6,8). Asegúrese de que para disolver completamente el pellet enjuagando las paredes de los tubos con una pipeta. </li><li> Incubar las muestras a 95 ° C durante 10 minutos y dejar enfriar a 20 ° C. </li><li> Centrifugar brevemente para recoger todo el líquido en la parte inferior de los tubos. </li><li> Cargue la cantidad total de cada muestra en los pocillos de un gel de poliacrilamida SDS (3.1.7). Utilice MOPS 50 mM, 50 mM Tris-X (Tris-base), EDTA 5 mM, 0,1% (w / v) SDS como tampón para electroforesis. </li><li>Ejecute el gel con 120 V durante aprox. 90 min. </li><li> Visualizar la fluorescencia en gel sobre una mesa de UV (312 nm) con una cámara CCD equipada con un filtro (540 nm ± 50 nm). NOTA: La luz UV es perjudicial para los ojos y la piel. </li></ol></li></ol>

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

Un paso etiquetado de ADN con MTases ADN y cofactores aziridina (ADN sonriendo) es un método robusto pero algunos aspectos se debe considerar al planear el experimento. Cofactor Aziridine: La concentración 6BAz para el etiquetado de ADN con M.BseCI fue de 60 mM. Cuando se utilizan otros MTases la concentración de ADN cofactor debe optimizarse, por ejemplo, concentraciones tan bajas como 20 mM han sido empleados con el ADN MTase M.TaqI 19. Las bajas concentraci...

Un etiquetado específico de los ácidos nucleicos y proteínas 1,2 3,4 es de gran interés para las caracterizaciones funcionales, diagnóstico médico y (nano) biotecnología. Aquí presentamos un método de marcaje enzimático para estos biopolímeros que se basa en S -adenosyl-l-metionina (AdoMet o SAM) metiltransferasas dependientes (MTases). Esta clase de enzimas (EC 2.1.1.) Se dirige a posiciones individuales nucleófilos (nitrógeno, oxígeno, azufre y átomos de carbono) dentro de los residuos específicos de ácidos nucleicos y proteínas y, naturalmente, transfiere el grupo metilo activado de la AdoMet cofactor (Figura 1A) 5. Además, MTases pueden utilizar análogos sintéticos cofactor para el etiquetado específico con etiquetas de afinidad, fluoróforos u otras etiquetas (Figura 1B) 6. Dos clases de análogos de AdoMet se han desarrollado: cofactores aziridina para concreto-equence S M ethyltransferase- I </strong&gt; Nduced L abel ing (sonriendo) 7 y dobles activados análogos AdoMet para m ethyltransferase dirigida TRANSFERENCIA DE UN ctivated rupos G (MTag) 8. <img alt="Figura 1" fo:content-width="7in" src="/files/ftp_upload/52014/52014fig1highres.jpg" width="700" /> Figura 1: reacciones catalizadas por metiltransferasas (MTases) A. Metil transferencia de grupo desde el cofactor natural de AdoMet (SAM) a diversos sustratos incluyendo ADN, ARN, proteínas y pequeñas biomoléculas B. Etiquetado / funcionalización de los ácidos nucleicos y proteínas (NNNNN =.. pares de bases de ADN, nucleótidos de ARN para ácidos y aminoácidos para las proteínas; XXXXX = secuencia de reconocimiento de la MTase con objetivo de residuos en verde) con análogos de cofactor sintéticos. Cofactores aziridina contienen un grupo indicador (azul esfera)unido al anillo de adenina son secuencia junto específicamente con el objetivo de residuos (izquierda) y de doble activado análogos de AdoMet llevar a la transferencia de cadenas de alquilo extendidos que llevan un reportero química Y (derecha) que puede ser etiquetado por reacción bioorthogonal clic en un segundo paso. <a href="https://www.jove.com/files/ftp_upload/52014/52014fig1highres.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> Cofactores aziridina funcionan mejor con ADN MTases. Contienen un anillo de tres miembros con un átomo de nitrógeno 9 (o un N de mostaza 10,11) en lugar del centro de sulfonio como grupo reactivo. La protonación de este átomo de nitrógeno activa el anillo de aziridina para el ataque nucleofílico por el nucleótido diana que conduce a Acoplamiento covalente de todo el cofactor con el ADN. Al conectar grupos informadores al anillo de adenina los cofactores de aziridina se pueden utilizar en combinación con ADN MTases para etiquetar ADN en un solo paso ( <stron<html> g&gt; Figura 1B, izquierda) 7,12. Esto se demuestra en detalle para la biotinilación de ADN con 6BAz 13-15 (cofactor de aziridina con biotina unido a la posición 6 del anillo de adenina) y la adenina específica de ADN MTase de Bacillus stearothermophilus (M.BseCI) 16 (Figura 2, véase la sección 2 del protocolo: un solo paso etiquetado de ADN a través de aziridina cofactores). Además de M.BseCI (&#39;secuencia de reconocimiento, la MTases ADN de Thermus aquaticus (M.TaqI, 5&#39;-TCG A -3 5&#39;-ATCG A T-3)&#39;), a partir de Haemophilus heamolyticus (M.HhaI, 5 &#39;-G C GC-3&#39;) y desde Spiroplasma (M.SssI, 5&#39;-C G-3 &#39;) se han utilizado con éxito para biotinilar ADN con 6BAz 17. Además, cofactores de aziridina pueden emplearse para un solo paso de fluorescencia de ADN etiquetado 18,19. ontenido &quot;fo: keep-together.within-page =&quot; always &quot;&gt; <img alt="Figura 2" fo:content-width="7in" src="/files/ftp_upload/52014/52014fig2highres.jpg" width="700" /> Figura 2:. Biotinilación específica de secuencia de un solo paso de ADN con M.BseCI y 6BAz El ADN MTase M.BseCI reconoce la secuencia de doble cadena de ADN 5&#39;-ATCG A T-3 &#39;y, naturalmente, metila el grupo amino de la segunda adenina residuo (verde) usando AdoMet. Con el cofactor aziridínico 6BAz el curso de la reacción se cambia y M.BseCI conduce a la secuencia de ADN específica biotinylation acoplando todo el cofactor incluyendo biotina (azul) con la adenina objetivo. <a href="https://www.jove.com/files/ftp_upload/52014/52014fig2highres.jpg" target="_blank">Por favor, haga clic aquí para ver una versión más grande de esta figura.</a> Doble análogos de AdoMet activados contienen extendieron cadenas laterales insaturadas en lugar de un grupo metilo en el centro de sulfonio (Figura 1B </strong&gt;, derecha) 20. El doble o triple enlace insaturado en β-posición al centro de sulfonio compensa electrónicamente efectos estéricos desfavorables en el estado de transición de la estabilización conjugativo. Dado que tanto el centro de sulfonio y el enlace insaturado activan la cadena lateral para la transferencia enzimática, estos cofactores fueron nombrados análogos AdoMet doble activados. Típicamente, se utilizan para transferir cadenas laterales con grupos únicos químicos (reporteros químicos), como los grupos amino, alquino y azida, para el etiquetado quimioselectivo en un segundo paso 8,21. En general, de doble activado análogos AdoMet puede no sólo funcionan como cofactores de ADN MTases 8,20,21 sino también para ARN MTases 22,23 y MTases proteínas 24-28 permitiendo etiquetado adicional de ARN y proteínas. Sin embargo, las cadenas laterales largos son estéricamente más exigente que un grupo metilo y ampliando las MTase sitios activos de proteínas de ingeniería es oftes necesario para obtener las velocidades de transferencia eficientes. Otra solución a este problema es utilizar un análogo AdoMet con un grupo pequeño de propargilo (tres carbonos) donde el alquino terminal sirve para dos funciones: 1. estabilización del estado de transición durante la transferencia enzimática y 2. mango reactiva para el seguimiento de las modificaciones químicas de cobre catalizada cicloadición-azida alquino (CuAAC) haga clic en la química. Resultó que la propargílico resultante AdoMet analógico 29 es bastante inestable en condiciones neutras o ligeramente básicas y sólo de uso limitado. Este inconveniente se puede solucionar mediante la sustitución del átomo de azufre con el selenio. El cofactor resultante 5 &#39;- [(Se) [(3 S) -3-amino-3-carboxipropil] prop-2-ynylselenonio] -5&#39;-desoxiadenosina (SeAdoYn, Figura 3) que es aceptado por el ADN de tipo salvaje, el ARN y MTases proteína 30-32 que abroga la necesidad de la ingeniería de proteínas en muchos casos. Esto se ejemplifica por fluorescencia pro<html> etiquetado proteína con la histona H3 lisina 4 (H3K4) MTase Set7 / 9 33 (Figura 3, véase la sección 3 del protocolo: el etiquetado de proteínas en dos pasos a través de cofactores dobles activados). <img alt="Figura 3" fo:content-width="7in" src="/files/ftp_upload/52014/52014fig3highres.jpg" width="700" /> Figura 3:. De dos pasos de fluorescencia etiquetado específico de secuencia de la histona H3 con Set7 / 9, SeAdoYn y TAMRA azida La proteína MTase Set7 / 9 metila naturalmente el grupo amino de la lisina 4 en la histona H3 (H3K4, verde) usando AdoMet. Con el cofactor de doble activado SeAdoYn la MTase transfiere un grupo propargilo pequeña (rojo) al residuo de lisina. El triple enlace terminal conectado es luego modificado selectivamente en un clic reacción bioorthogonal (azida alquino cicloadición catalizada por cobre, CuAAC) con (tetrametilrodamina, azul) fluoróforo azida derivatizado TAMRA.carga / 52014 / 52014fig3highres.jpg &quot;target =&quot; _ blank &quot;&gt; Haga clic aquí para ver una versión más grande de esta 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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<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

S -Adenosyl-L-metionina (AdoMet o SAM) metiltransferasas dependientes (MTase) catalizan la transferencia del grupo metilo activado a partir de AdoMet a posiciones específicas en el ADN, ARN, proteínas y pequeñas biomoléculas. Esta reacción de metilación natural puede ampliarse a una amplia variedad de reacciones de alquilación utilizando análogos cofactor sintéticos. La sustitución del centro de sulfonio reactiva de AdoMet con un anillo de aziridina conduce a cofactores que se pueden acoplar con el ADN por varios MTases de ADN. Estos cofactores de aziridina pueden ser equipados con grupos informadores en diferentes posiciones del resto de adenina y se utilizan para S-equence específica M ethyltransferase- I nduced L Abel ING de ADN (DNA sonriendo). Como un ejemplo típico damos un protocolo para la biotinilación de ADN del plásmido pBR322 en la secuencia 5&#39;-ATCG A T-3 &#39;con el MTase M.BseCI ADN y el cofactor aziridina en 6BAzun solo paso. Extensión del grupo metilo activado con grupos alquilo insaturados resultados en otra clase de análogos de AdoMet que se utilizan para m ethyltransferase dirigida TRANSFERENCIA DE UN ctivated rupos G (MTag). Dado que las cadenas laterales largos son activados por el centro de sulfonio y el enlace insaturado, estos cofactores son llamados análogos de AdoMet doble activadas. Estos análogos no sólo funcionan como cofactores para MTases de ADN, como los cofactores de aziridina, sino también para el ARN, proteínas y pequeños MTases molécula. Se utilizan típicamente para la modificación enzimática de sustratos MTase con grupos funcionales únicos que se etiquetan con grupos informadores en una segunda etapa química. Esto se ejemplifica en un protocolo para el marcaje de fluorescencia de la proteína histona H3. Un grupo propargilo pequeña se transfiere desde el SeAdoYn análogo cofactor de la proteína por la histona H3 lisina 4 (H3K4) MTase Set7 / 9 seguido por clic etiquetado de lahistona H3 alkynylated con TAMRA azida. Etiquetado mediada MTase con análogos cofactor es una tecnología que permite muchas aplicaciones interesantes, como la identificación y el estudio funcional de sustratos MTase así como el genotipado de ADN y detección de metilación.

Un paso Etiquetado de ADN a través de aziridina cofactores Esta reacción se lleva a cabo ejemplo con el ADN MTase M.BseCI, que se modifica el segundo residuo de adenina en el extremo 5 &#39;ATCG A T-3&#39; secuencia de doble cadena y tiene un sitio de reconocimiento en el plásmido pBR322 (Figura 4A). Para probar etiquetado plásmido, pBR322 es desafiado con la endonucleasa de restricción (REase) R.TaqI (5&#39;-TCGA-3 &#39;). R.TaqI tiene siete s...

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

ADN y las proteínas se marcan secuencia específicamente con afinidad o grupos indicadores fluorescentes usando ADN o de proteínas metiltransferasas y análogos sintéticos de cofactor. Dependiendo de la especificidad de cofactor de las enzimas, aziridina o dobles análogos de cofactores activados se emplean para el etiquetado de uno o dos pasos.

Etiquetado específico de secuencia de ácidos nucleicos y proteínas con metiltransferasas y el cofactor Análogos

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 vistas.  RWTH Aachen University.  ADN y las proteínas se marcan secuencia específicamente con afinidad o grupos indicadores fluorescentes usando ADN o de proteínas metiltransferasas y análogos sintéticos de cofactor. Dependiendo de la especificidad de cofactor de las enzimas, aziridina o dobles análogos de cofactores activados se emplean para el etiquetado de uno o dos pasos. 

Video: 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.

Detección por inmunofluorescencia de dos análogos de la timidina (CldU y UDI) en los tejidos de Primaria

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

Investigación

Biología

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.

Perfiles de metiltransferasas y Otros S-Adenosil- L-Homocisteína proteínas que se unen mediante espectrometría de masas de captura compuesto (MCP)

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 microvolumen cuantificación de ácidos nucleicos

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.

Examinar la dinámica conformacional de las proteínas de membrana In situ Sitio-dirigida con fluorescencia de etiquetado

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.

Etiquetado linaje de células de pez cebra con láser Uncagable fluoresceína 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.

Determinación de la concentración de ácidos nucleicos y proteínas utilizando el micro-volumen Bio-spec Espectrofotómetro 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 etiquetado y análisis de imágenes de las proteínas en las células del sensor 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.

Confirmación vía metabólica y el descubrimiento a través de 13 C-etiquetado de proteinogenic 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.

Célula de medida Cinética la progresión del ciclo con el etiquetado metabólico y Citometría de Flujo

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.

Análisis y construcción de estructuras de ácidos nucleicos con 3DNA

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