La asistencia técnica y la instrumentación de la Universidad de Maryland Fondo para el Centro de Cáncer Greenebaum Traslacional Core, especialmente los Dres. Rena Lapidus y Mariola Sadowska, se agradece. El trabajo responsable de la elaboración de este ensayo fue financiado en parte por el NIH RO1CA108846, AHA Grant-in-Aid GRNT2130014, un Premio al Mérito VA a AP, y por la Universidad de Maryland de cigarrillos fondos de reembolso (CRF) proporcionada a la Marlene y Stewart Centro del Cáncer de Greenbaum.

Varios pasos se llevan a cabo antes de tiempo y varios reactivos se preparan y se almacenan antes del procedimiento: (1) cultivo de células y proteínas de aislamiento, (2) preparación de oligonucleótido, (3) la preparación de placas de 96 pocillos, (4) extracto nuclear durante la noche de incubación. Procedimiento requiere 2 días debido a la incubación durante la noche de la proteína nuclear con oligonucleótidos de ADN. 1. Preparación de Buffers 1.1 aislamiento de proteínas nucleares <ol><li> Hipotónico Buffer: Para preparar 100 ml de tampón hipotónico, añadir 1 ml (1 M HEPES, pH 7,9), 150 l (1 M MgCl 2), 333 l (3 M KCl) a 98,3 ml de H 2 O. Las concentraciones finales: 10 mM HEPES, 1,5 mM de MgCl 2, 10 mM de KCl. Para tampón hipotónico + NP40 (para su uso en el paso 2.11): Añadir 0,5 ml (10% NP40) a 9,5 ml de tampón hipotónico final de 0,5% NP40. </li><li> Buffer Bajo en sal: Para preparar 100 ml de tampón de sal baja (para el uso en el paso 2.15), añadir 1 ml (1 M HEPES, pH 7,9), 25 ml de glicerol, 150 l (1 M MgCl 2), 666 l (3 M KCl), 40 l (0,5 M EDTA) a 73 ml de H 2 O. Las concentraciones finales: 10 mM de HEPES, 25% de glicerol, 1,5 mM de MgCl 2, KCl 20 mM, EDTA 0,2 mM. </li><li> Salinidad Buffer: Para preparar 100 ml de tampón de sal de alta (para el uso en el paso 2.15), añadir 1 ml (1 M HEPES, pH 7.9), 25 ml de glicerol, 150 l (1 M MgCl 2), 26,7 ml (3 M KCl), 40 l (0,5 M EDTA) a 47 ml de H 2 O. Las concentraciones finales: 10 mM de HEPES, 25% de glicerol, 1,5 mM de MgCl 2, 800 mM de KCl, 0,2 mM de EDTA. </li><li> 10% de NP40 Detergente: 10 ml de NP40 a 90 ml de H2O </li><li> Los inhibidores de proteasa y fosfatasa [añadido al tampón hipotónico + NP40 (paso 2,11), al tampón de baja sal (paso 2,15), y para tampón de alto contenido de sal (paso 2,15) justo antes de su uso]: Añadir 2,5 l (1 M DTT) y 50 l de inhibidores de la proteasa (100x) a 5 ml de cada tampón para concentración final de 0,5 mM de DTT y los inhibidores de la proteasa 1x. El inhibidor de la proteasa mezcla 100x valores incluye:fluoruro de sodio (Ser / Thr, fosfatasas ácidas), ortovanadato de sodio (Tyr, fosfatasas alcalinas), β-glicerofosfato (fosfatasas Ser / Thr), pirofosfato de sodio (fosfatasas Ser / Thr), aprotinina (Ser proteasas), bestatina (amino-peptidasas ), E64 (proteasas de cisteína), leupeptina (proteasas Ser / Cys), EDTA (metaloproteasas). </li></ol> 1.2 Preparación de placas de ensayo <ol><li> Placa de fijación de solución: Para preparar 500 ml de solución, añadir 5,25 g de carbonato de sodio a 500 ml de H2O, mezclar bien y ajustar el pH a 9,7 para una concentración final de carbonato de sodio 0,1 M. </li><li> Placa de solución de bloqueo: Para preparar Stock poli dI / DC oligonucleótido, resuspender 1 Unidad (~ 50 mg) de poli dI / DC en 1 ml de 10 mM de Tris / HCl, pH 7,9 que contiene EDTA 1 mM para una concentración de solución madre de 50 mg / l. El stock se diluye a 1 g / l antes de su uso. </li></ol> 1.3 tampones de lavado y diluciones de anticuerpos NOTA: tampón de lavado estreptavidina es<html> utilizado para lavar platos en entre adiciones y para diluir los anticuerpos primarios y secundarios. <ol><li> Para preparar 1 l de tampón de lavado estreptavidina, añadir 2,4 g de Tris / HCl, 37,5 ml (4 M de NaCl), 10 ml (10% de BSA), 5 ml (10% de Tween-20), a 1 L de agua filtrada Millipore estéril , pH 7,18. Almacenar a 4 ° C. Las concentraciones finales: 20 mM de Tris / HCl, NaCl 150 mM, 0,1% de BSA, 0,05% de Tween-20. </li></ol> Tampón de unión a ADN 1,4 NOTA: Este tampón se almacenó a -20 ° C. <ol><li> Para preparar 15 ml de tampón de unión de ADN, añadir 180 l (1 M HEPES, pH 7,9), 300 l (3 M de KCl), 12 l (0,5 M EDTA, pH 8,0), 7,5 l (1 M DTT), 3,0 ml (60% de glicerol), 300 l (50 g / l de poli dI / DC) diluido con agua desionizada / destilada H 2 O (12 ml). Las concentraciones finales: 12 mM HEPES / pH 7,9, 60 mM KCl, EDTA 0,4 mM, DTT 0,5 mM, 12% de glicerol, 1 g / l de poli dI / DC. </li></ol> 2. Cultivo de células y aislamiento de proteínas nucleares t &quot;&gt; NOTA: La preparación requiere alrededor de 3 horas de estimulación de las células dependerá de experimento El número de células utilizadas para cada experimento puede variar y todos los volúmenes refleja un experimento típico utilizando 3 x 10 7 células / punto de ajustar los volúmenes de acomodar... el número de células realmente utilizada en el experimento 6. <ol><li> Para preparar la proteína nuclear para los ensayos de unión de ADN, células humanas de cultivo que expresan RUNX2 (endotelial, osteosarcoma, células de cáncer de mama) en los medios de comunicación adecuados 6. </li><li> Tratar las células subconfluentes con nocodazole (0,2 mg / ml durante 16 horas) para detener las células en el ciclo celular límite G2 / M 6. En estas condiciones, la proteína RUNX2 se estabiliza y se consigue la unión de ADN máxima. De esta manera, suficiente proteína nuclear está disponible para múltiples ensayos y los inhibidores se pueden comparar a partir de la misma preparación. </li><li> Para cada paso, pre-enfriar la centrífuga a 4 ° C y preparar tampones y enfriar 20 min a 4 ° C antes de la cosecha de células.</li><li> Células Chill (4 ° C) y procedimientos de conducta en el hielo. </li><li> Centrifugar las células a 1000 rpm durante 5 min en un tubo de fondo redondo de 15 ml de polipropileno. </li><li> Lavar 1x con helado de PBS (Ca 2 + y Mg 2 + libre). </li><li> Se centrifuga de nuevo y desechar PBS sobrenadante. </li><li> Añadir 500 l de tampón hipotónico y sin inhibidores de la proteasa, teniendo cuidado para resuspender completamente el sedimento celular. </li><li> Transferir contenido a un tubo Eppendorf de 1,5 ml. </li><li> Inmediatamente centrifugar a 5.500 rpm durante 5,5 min; descartar el sobrenadante y mantener el sedimento celular. </li><li> Resuspender el sedimento de células en 500 l de tampón hipotónico que contienen 0,5% de NP40. Añadir la proteasa y los inhibidores de la fosfatasa y DTT 0,5 mM (tal como se describe en el paso 1.1.5). </li><li> Permitir que la muestra para descansar en hielo durante 30 min. </li><li> Centrifugar a 13000 rpm durante 10 min. </li><li> Descartar el sobrenadante (que contiene las proteínas citosólicas). </li><li> Resuspender el botón nuclear en 60 l cada uno de baja salt tampón (a la que los inhibidores de la proteasa y DTT 0,5 mM, se han añadido de la etapa 1.1.5) y el tampón de sal alta (a la que los inhibidores de la proteasa y DTT 0,5 mM, se han añadido de la etapa 1.1.5) para un total de 120 l. Añadir 60 l de tampón de baja salinidad al sedimento primero y volver a suspender, a continuación, añadir 60 l de tampón de alta salinidad. Agite el pellet para ayudar en la resuspensión. </li></ol> NOTA: Este paso ayuda a deshacerse de algunas de las proteínas de la membrana nuclear y prepara para el paso de lisis: baja en sal primero (pellet se resuspende, vórtice), a continuación, alto contenido de sal (vortex) optimiza este paso. Mantener extracto en hielo durante 30 min, con agitación con vórtex después de la primera 10 min. <ol start="16"><li> Centrifugar a 12000 rpm durante 15 min; mantener el sobrenadante que contiene la proteína nuclear. </li><li> Medir la concentración de proteínas (trate de mantener las muestras a 2-5 mg / ml), la alícuota, en cantidades adecuadas (10 l / tubo) y se almacena a -80 ° C. </li></ol> NOTA: El rendimiento estimado: 30 x 10 6 celLS producirán 5 g / l de proteína en 120 l. 5 x 10 6 células producirán 2,2 g / l de proteínas en 40 l. 3. Preparación de la doble cadena de oligonucleótidos <ol><li> Diseñar oligonucleótidos complementarios con los sitios de media compatibles en los extremos. Para RUNX2 estos son: 5&#39;-CGTATT AACCACA ATACTCGCGTATT AACCACA ATACTCGCGTATT AACCACA ATAC TCG-3&#39;-biotina (sentido) y 5&#39;CGAGTAT TGTGGTT AATACGCGAGTAT TGTGGTT AATACGCGAGTAT T GTGGTT AATACG-3&#39;-biotina (antisentido). </li></ol> NOTA: Los experimentos piloto determinó que tres sitios de unión RUNX2 y single-end marcado con biotina produjeron los resultados más reproducibles. <ol start="2"><li> Prepare el tampón de hibridación 5x: Añadir 300 l (1 M Tris-HCl, pH 7,6), 30 l (1 M MgCl 2), 10 l (1 M DTT) a 260 l H 2 O (finales de 600 l). </li><li> Para 200 pmol de cada olig monocatenarioonucleotide (3 l), añadir 12 l de tampón de recocido 5x y 45 l H 2 O para un total de 60 l (200 pmol/60 mu l). </li><li> Se calienta a 95 ° C durante 5-10 min en un bloque de calentamiento. </li><li> Enfriar a 65 ° C lentamente (ajustando la temperatura a 65 ° C, se tarda 15 min), luego se enfría a temperatura ambiente lentamente de desconectar la alimentación del bloque de calentamiento (o colocando en 50 ml de agua a 65 ° C en un vaso de hielo, lo que tarda 15-20 minutos). </li></ol> 4. Preparación de placas de 96 pocillos NOTA: No utilizar proteínas de la leche en los tampones de lavado. <ol><li> Fijar la placa con solución de fijación (0,1 M de carbonato de sodio): añadir 300 l / pocillo. </li><li> Incubar durante 2 horas, meciendo a temperatura ambiente (RT), o ninguna mecedora a 4 ° C (O / N). </li><li> Lave la placa 3x con tampón de lavado estreptavidina (BM): añadir 300 l / pocillo cada vez. </li><li> Añadir oligonucleótido de doble cadena marcado con biotina (3 sitios de unión de consenso por nucleótido) durante 2 horas wi-ésimo de balanceo (1,25 nmol / pocillo; 100 l / pocillo). </li><li> Lave 3 veces con frío WB: añadir 300 l / pocillo y luego invertir inmediatamente la placa en un fregadero y borra los bordes en una toalla de papel después de cada adición. </li></ol> NOTA: No utilice las puntas de pipeta para extraer líquido de las placas ya que esto puede raspar avidina: biotina: ADN de los pozos. Haga esto para todas las etapas de lavado posteriores. 5. La incubación con extracto nuclear NOTA: No sustituya el salmón o el ADN de esperma de arenque para el poli tampón de bloqueo dI / DC. Evite bloquear las placas con proteínas de la leche. Ambos de estos agentes de bloqueo resultan en altos valores de fondo. <ol><li> Incubar con factor de transcripción específico preparado a partir de extracto nuclear (90 l / pocillo). Preparar una mezcla maestra que contiene extracto nuclear (proteína de unión a ADN, 3-9 mg / pocillo) + poli dI / DC (1 g / l de un mg / Stock ul 50) en el ADN de 1x tampón de unión. El volumen se como sea necesario para el número total depozos requeridos (90 l / pocillo). </li><li> Cualquier inhibidor potencial, tales como la vitamina D3 (Figura 2) o compuesto CADD 5221975 (Figura 3), o la dilución apropiada del control de disolvente, tal como etanol, se añade en este paso. </li><li> Colocar la placa en la plataforma oscilante, O / N a 4 ° C. </li><li> Lave 3x con WB: añadir 300 l / pocillo </li></ol> 6. La adición de Anticuerpo Primario NOTA: diluciones de anticuerpos primarios deben estar preparados frescos - No se recomienda el almacenamiento. <ol><li> Diluir el anticuerpo monoclonal-RUNX2 específica (acción 1 mg / l) 1/5, 000 en estreptavidina Tampón de lavado. Preparar suficiente para la adición a cada pocillo de la placa de 96 pocillos (90 l / pocillo) por triplicado. </li><li> Incubar durante 1 hora a temperatura ambiente en una plataforma oscilante. </li><li> Lave 3x con WB, añadir 300 l / pocillo. </li></ol> 7. La adición de anticuerpo secundario NOTA: diluciones de anticuerpos secundarios pueden ser storojo a 4 ° C durante la noche si es necesario el día siguiente. <ol><li> Diluir F purificado por afinidad-HRP anticuerpo conjugado (7,1 mg / ml) ab-específica a 1:1,400 en estreptavidina Tampón de lavado: tampón de lavado ml 3,5 l antibody/5.0. Preparar suficiente para la adición a cada pocillo de la placa de 96 pocillos (90 l / pocillo) por triplicado. </li><li> Incubar durante 30 min a temperatura ambiente en una plataforma oscilante. </li><li> Lave 6x con WB invirtiendo la placa en el fregadero (300 l / pocillo). </li></ol> 8. HRP Sustrato y Desarrollo de Productos <ol><li> Añadir 50 l de sustrato TMB por pocillo directamente desde el almacén botella. </li><li> Incubar 10-20 minutos a temperatura ambiente en la oscuridad (parada método de reacción) o medir la absorción directa (monitoreo cinética continua). </li></ol> 9. Reacción Medición <ol><li> Deje de método de reacción: Para la reacción de parada visual, compruebe el cambio de color de claro a azul y detener la reacción con ácido sulfúrico 50 l. </li><li> Medir la absorbancia a 450 nm conel espectrofotómetro Biotrak II lector de placa visible (Amersham Biosciences; GE Healthcare Biosciences Corp. Piscataway NJ, EE.UU.) o un instrumento similar. </li><li> Continuo método de monitoreo cinética: Añadir sustrato después del último lavado y justo antes de colocar en el espectrofotómetro (no detener la reacción). </li><li> Medir la absorbancia a 635 nm con el Bio-Tek Sinergia HT multi-reacción lector de microplacas Espectrofotómetro (Bio-Tek Instruments, Inc; Winooski, VT, EE.UU.) o un instrumento similar. </li></ol>

<ol>
	<li>Hellman, L. M., Fried, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+mobility+shift+assay+(EMSA)+for+detecting+protein-nucleic+acid+interactions.">Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions.</a> Nat. Protoc. 2, 1849-1861 (2007).</li><li>Bhattacharya, N., Sarno, A., Idler, I. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+detection+of+nuclear+factor-kappaB+activity+using+a+sensitive+oligo-based+chemiluminescent+enzyme-linked+immunosorbent+assay.">High-throughput detection of nuclear factor-kappaB activity using a sensitive oligo-based chemiluminescent enzyme-linked immunosorbent assay.</a> Int. J. Cancer. 127, 404-411 (2010).</li><li>Hartzell, D. D., Trinklein, N. D., Mendez, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+analysis+of+the+CREB+signaling+pathway+using+HaloCHIP-chip+and+high+throughput+reporter+assays.">A functional analysis of the CREB signaling pathway using HaloCHIP-chip and high throughput reporter assays.</a> BMC Genomics. 10, 497 (2009).</li><li>Vuori, K. A., Ahlskog, J. K., Sistonen, L., Nikinmaa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TransLISA,+a+novel+quantitative,+nonradioactive+assay+for+transcription+factor+DNA-binding+analyses.">TransLISA, a novel quantitative, nonradioactive assay for transcription factor DNA-binding analyses.</a> FEBS J. 276, 7366-7374 (2009).</li><li>D'Souza, D. R., Salib, M. M., Bennett, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperglycemia+Regulates+RUNX2+Activation+and+Cellular+Wound+Healing+through+the+Aldose+Reductase+Polyol+Pathway.">Hyperglycemia Regulates RUNX2 Activation and Cellular Wound Healing through the Aldose Reductase Polyol Pathway.</a> J. Biol. Chem. 284, 17947-17955 (2009).</li><li>Underwood, K. F., D'Souza, D. R., Mochin, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+RUNX2+transcription+factor-DNA+interactions+and+cell+proliferation+by+Vitamin+D3+(cholecalciferol)+prohormone+activity.">Regulation of RUNX2 transcription factor-DNA interactions and cell proliferation by Vitamin D3 (cholecalciferol) prohormone activity.</a> J. Bone Miner Res. 27, 913-925 (2012).</li><li>Held, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+ß-galactosidase+activity+using+the+PowerWave+HT+and+Gen5+data+analysis+software:+basic+enzyme+kinetic+determinations.">Kinetic analysis of ß-galactosidase activity using the PowerWave HT and Gen5 data analysis software: basic enzyme kinetic determinations.</a> Biotek Instruments Application Note. , (2007).</li><li>Renard, P., Ernest, I., Houbion, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+sensitive+multi-well+colorimetric+assay+for+active+NFkappaB.">Development of a sensitive multi-well colorimetric assay for active NFkappaB.</a> Nucleic Acids Res. 29, E21 (2001).</li><li>Pommier, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+topoisomerase+I+inhibitors:+chemistry,+biology,+and+interfacial+inhibition.">DNA topoisomerase I inhibitors: chemistry, biology, and interfacial inhibition.</a> Chem Rev. 109, 2894-2902 (2009).</li><li>Pennisi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomics.+ENCODE+project+writes+eulogy+for+junk+DNA.">Genomics. ENCODE project writes eulogy for junk DNA.</a> Science. 337, 1159-1161 (2012).</li></ol>

No tenemos nada que revelar.

Ensayos de unión al ADN se utilizan para medir la capacidad de los factores de transcripción para interactuar con el ADN. Los ensayos para la unión del ADN incluyen cambio de la movilidad electroforética (EMSA) y 1 inmunoprecipitación de la cromatina (ChIP) ensayos basados ​​3, así como ensayos que emplean formatos de 96 pocillos 4 tales como ensayos quimioluminiscentes 2. La EMSA es no cuantitativa y utiliza radiomarcado (32 P) oligonucleótidos. Estos se...

Ensayos de unión al ADN tienen utilidad en la medición de la capacidad de los factores de transcripción para interactuar con el ADN. Los ensayos para ADN de unión incluyen ensayos de cambio de movilidad electroforética (EMSA) que dependen de oligonucleótidos radiomarcados 1 o ensayos de quimioluminiscencia 2. Ensayos de inmunoprecipitación de la cromatina (ChIP) basadas 3, así como ensayos que emplean formatos de 96 pocillos 4 también se han descrito. Sin embargo, la EMSA es un ensayo de no cuantitativa que requiere el uso de oligonucleótidos radiomarcados. Cuando las proteínas nucleares se asocian con las secuencias específicas de nucleótidos promotora, complejos de unión están retrasados ​​en geles de poliacrilamida y el factor de transcripción específico puede ser validado con una &quot;supershift&quot; anticuerpo. Hemos desarrollado un ensayo de unión de ADN-cuantitativa utilizando un formato de inmunoabsorción ligado a enzimas (D-ELISA), que es capaz de medir la interacción de RUNX2 con secuencias de unión al ADN que corresponden a elementos promotores definidos igenes objetivo n RUNX2. El uso de un anticuerpo anti-RUNX2 proporciona especificidad para el ensayo y la falta de radiomarcador distinguir este ensayo a partir del ensayo de desplazamiento en gel tradicional 5. La detección de complejos de unión es posible con el uso de un anticuerpo secundario acoplado a peroxidasa de rábano picante (HRP), que convierte un sustrato de HRP, tetrametil bencidina (TMB) en un producto coloreado para el análisis espectrofotométrico. El ensayo informado aquí puede incorporar el uso de oligonucleótidos de ADN mutadas como controles y se puede utilizar para la detección de inhibidores competitivos o no competitivos de la unión del ADN. La selección de compuestos antitumorales novedosos también es posible con este ensayo.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the Reagent</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Poly dI/dC</td>
			<td>GE Healthcare, Piscataway, NJ</td>
			<td>US20539-5UN</td>
			<td>1 U ~50mg</td>
		</tr>
		<tr>
			<td>RUNX2 antibody</td>
			<td>MBL International Corp., Woburn, MA</td>
			<td>D130-3</td>
			<td>1 mg/ml</td>
		</tr>
		<tr>
			<td>Fab-specific peroxidase conjugated antibody</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>A9917</td>
			<td>7.1 mg/ml</td>
		</tr>
		<tr>
			<td>TMB Substrate (tetramethyl benzidine)</td>
			<td>EXALPHA Biologicals, Shirley, MA</td>
			<td>X1189S</td>
			<td>100 ml</td>
		</tr>
		<tr>
			<td>Sodium carbonate</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>57995</td>
			<td>Plate-fixing</td>
		</tr>
		<tr>
			<td>Sulfuric Acid</td>
			<td>VWR, West Chester, PA</td>
			<td>BDH-39922-1</td>
			<td>Stop solution</td>
		</tr>
		<tr>
			<td>Multi-well plates</td>
			<td>Greiner Bio-One, Basel, Switzerland</td>
			<td>655996</td>
			<td>Avidin-coated, black sides</td>
		</tr>
		<tr>
			<td>HALT</td>
			<td>Thermo-Scientific/Pierce, Rockford, IL</td>
			<td>78440</td>
			<td>Protease and phosphatase inhibitors</td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td>Various manufacturers</td>
			<td>Laboratory grade</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Table 1. Reagents</td>
		</tr>
		<tr>
			<td>Spectrophotometer: Biotrak II Visible plate reader</td>
			<td>Amersham Biosciences</td>
			<td>&nbsp;</td>
			<td>For use with stop reaction method</td>
		</tr>
		<tr>
			<td>Spectrophotometer: Bio-Tek Synergy HT Multi-reaction microplate reader</td>
			<td>Bio-Tek Instruments, Inc.</td>
			<td>&nbsp;</td>
			<td>For use with continuous kinetic monitoring</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Table 2. Equipment</td>
		</tr>
	</tbody>
</table>

a quantitative assay to study protein:dna interactions, discover transcriptional regulators of gene expression, and identify novel anti-tumor agents

Muchos ensayos de unión de ADN-tales como ensayos de cambio de movilidad electroforética (EMSA), ensayos de quimioluminiscencia, de inmunoprecipitación de la cromatina (ChIP) a base de ensayos, y ensayos basados ​​en pocillos múltiples se utilizan para medir la actividad del factor de transcripción. Sin embargo, estos ensayos son no cuantitativos, carecen de especificidad, pueden implicar el uso de oligonucleótidos radiomarcados, y pueden no ser adaptables para el cribado de inhibidores de la unión del ADN. Por otro lado, el uso de una unión a ADN ensayo cuantitativo inmunoabsorbente ligado a enzimas (D-ELISA), que demuestran las interacciones de proteínas nucleares con ADN utilizando el factor de transcripción RUNX2 que dependen de asociación específica con secuencias de unión al ADN consenso presentes en biotina- oligonucleótidos marcados. Preparación de las células, la extracción de proteína nuclear, y el diseño de oligonucleótidos de doble cadena se describen. Placas de 96 pocillos recubiertas con avidina se fijan con tampón alcalino y se incubaron con las proteínas nucleares en tampón de bloqueo de nucleótidos. Folldebido extenso lavado de las placas, el anticuerpo primario específico y las incubaciones de anticuerpos secundarios son seguidos por la adición de sustrato de peroxidasa de rábano picante y el desarrollo de la reacción colorimétrica. Modo de reacción de parada o un control cinético continuo se utilizaron para medir cuantitativamente la interacción de proteínas con el ADN. Se discuten controles de especificidad apropiadas, incluyendo el tratamiento con IgG no específica o sin proteína o anticuerpo primario. Aplicaciones del ensayo se describen incluyendo su utilidad en la detección de drogas y se discuten los resultados positivos y negativos representativos.

El método D-ELISA es altamente específico para la proteína de unión a ADN designado, siempre y cuando se utiliza un oligonucleótido de doble cadena específica de la secuencia que contiene tres copias del sitio de unión consenso RUNX2 (ACACCA). El anticuerpo primario que reconoce el factor de proteína también aumenta la especificidad. El anticuerpo secundario contiene covalentemente unido a la peroxidasa de rábano picante (HRP) que convierte un sustrato transparente (tetrametil bencidina) en un producto colorea...

Watch this Scientific Journal Video about A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents at JoVE.c

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Desarrollamos una unión a ADN, ensayo basado en ELISA cuantitativo para medir las interacciones de los factores de transcripción con el ADN. Alta especificidad para la proteína RUNX2 se logró con un oligonucleótido de ADN consenso de reconocimiento y el anticuerpo monoclonal específico. Detección colorimétrica con una reacción anticuerpo-enzima-sustrato acoplado se monitorizó en tiempo real.

Un ensayo cuantitativo para estudiar proteínas: DNA Interacciones, reguladores Descubra transcripcional de la expresión génica, e identificar agentes antitumoral

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

a-quantitative-assay-to-study-proteindna-interactions-discover

Research

JoVE Journal

Biology

18.5K vistas.  University of Maryland School of Medicine.  Desarrollamos una unión a ADN, ensayo basado en ELISA cuantitativo para medir las interacciones de los factores de transcripción con el ADN. Alta especificidad para la proteína RUNX2 se logró con un oligonucleótido de ADN consenso de reconocimiento y el anticuerpo monoclonal específico. Detección colorimétrica con una reacción anticuerpo-enzima-sustrato acoplado se monitorizó en tiempo real. 

Video: A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

La preparación de cultivos primarios de células hematopoyéticas de la médula ósea murina por electroporación

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Investigación

Biología

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Utilizando el sistema de electroporación Gene Emisores MXcell para transfectar células primarias con alta eficiencia

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

Hi-C: Un método para estudiar la arquitectura tridimensional de los genomas.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

El uso de un contador de células automatizado para simplificar estudios de expresión génica: siRNA Derribo de IL-4 la expresión génica en células dependientes Namalwa

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

Inmunoprecipitación de cromatina (CHIP) para el ensayo dinámico de modificación de las histonas en la expresión génica activado en las células humanas

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

En la reconstitución in vitro de los activos T. castaneum telomerasa

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

El aislamiento de los ribosomas polipéptidos nacientes Bound<em&gt; In vitro</em&gt; Para la identificación de sitios de traslación pausa a lo largo del ARNm

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Análisis de RNA Global Síntesis en el nivel de células individuales siguiente Hipoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Screening siRNA para identificar ubiquitina y reguladores del sistema ubiquitina-como de las vías biológicas en células de mamífero en cultivo

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

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

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

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

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

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