Este trabajo fue financiado por los Institutos Canadienses de Investigación en Salud (CIHR), Nacional de Ciencias e Ingeniería de Investigación (NSERC), y una beca de estudios de formación CIHR (a LF). Agradecemos al Prof. Gerard D. Wright (Universidad de McMaster, Canadá) para la AAC (6)-Ii plásmido de expresión.

1. Preparación de soluciones madre <ol><li> Purificar la macromolécula de interés. (En este caso, aminoglucósidos N-6&#39;-acetil-II (AAC6&#39;-II), se encuentra aislada como se informa en otro lugar. 13) </li><li> Preparar 4 litros de tampón de diálisis. (En este caso se utilizó 25 mM 4 - (2-hidroxietil)-1-piperazineethanesulfonic ácido (HEPES, 238,3 MW g / mol), que contiene 2 mM de ácido etilendiaminotetraacético (EDTA, 292,2 MW), a pH 7,5.) </li><li> Dializar la proteína de la AAC (6 &#39;)-II muestra (5 ml a 400 M) deben ser dializados en el tampón de diálisis (3 x 1,3 L). La solución final de diálisis se reduce al lavado de la máquina y para diluir las muestras. </li><li> Filtrar la solución final de diálisis a través de un filtro de 0,45 micras de celulosa, y se almacenan a 4 ° C. Se conoce como &#39;buffer corriente &quot; </li><li> Solución de proteína filtra a través de un filtro de 0,2 micras jeringa que ha sido lavada a fondo con buffer en funcionamiento. Medir la concentración final de la proteína utilizando un ensayo estándar (Bradford, Lowry, etc) o de absorción UV. Tienda de la proteína para maximizar la estabilidad a largo plazo. (En el caso de la AAC (6 &#39;)-II, este medio de almacenamiento a 4 ° C. AAC (6&#39;)-II no retiene la actividad después de la congelación y descongelación). </li><li> Prepare 200μL de solución madre 25 mM de acetil coenzima A (AcCoA, el ligando, 809,57 MW) mediante la disolución de 4,0 mg en la gestión de buffer. Se congelan a -78 º C hasta su utilización. </li><li> Todas las muestras de proteínas y ligandos deben proceder de las soluciones misma población, para minimizar la muestra aleatoria de la muestra de las fluctuaciones en la concentración. Un factor de corrección único para la concentración de proteínas en todo el conjunto de datos c variable se ajusta en el análisis 10. </li></ol> 2. Preparación de muestras de ITC <ol><li> Diluir la solución de la enzima a un valor de c de 64 años con un volumen final de 2 ml. (En el caso de la AAC (6 &#39;)-II, lo que corresponde a 192 M). </li><li> Rápido deshielo del ligando (AcCoA) solución de reserva de agua helada. </li><li> Preparar 0.5 mL de una solución AcCoA 10 veces más concentrado y luego el número de sitios de unión de la proteína, en este caso 4 mM, mediante la dilución de 80 l de la solución madre AcCoA en 420 l de funcionamiento de amortiguación. Transferir a un tubo de llenado de la pipeta. Rápido retorno de la solución madre AcCoA a -78 ° C. </li><li> Degas la proteína y las soluciones al vacío durante 5 minutos a una temperatura de 1 ° C por debajo de la temperatura deseada en funcionamiento (19 ° C). </li></ol> 3. La creación de la jeringa 14 <ol><li> Inserte la jeringa a través de soporte de jeringa hasta que la abrazadera jeringa montada es a la misma altura que el titular. </li><li> Alimentar a la pinza de segunda jeringa en la jeringa hasta que se presiona firmemente contra la parte inferior del soporte de jeringa. Apriete suavemente la pinza con la proporcionada 0.050 &quot;Drive bola hexagonal Point. </li><li> Coloque la jeringa titular en el soporte de la pipeta. </li><li> Deslice con cuidado la pipeta en el inyector de jeringa de inyección. Asegúrese de que el émbolo se introduce directamente en el agujero de la jeringa. Una vez insertado, el tornillo del collar de bloqueo del titular de la jeringa en el inyector de la pipeta. </li></ol> 4. Carga de la célula de la muestra 14 <ol><li> Lave la celda de muestra con un mínimo de 50 ml de funcionamiento de amortiguación, y eliminar todo el líquido restante con una larga aguja 2,5 ml jeringa de vidrio. </li><li> Poco a poco sacar un mínimo de 1,8 ml de solución de proteína en la muestra limpia y seca de larga aguja jeringa de 2,5 ml. Tenga cuidado de no introducir burbujas. </li><li> Con cuidado, inserte la aguja en la celda de la muestra y tocar suavemente la parte inferior de la celda. Levante la punta ligeramente (~ 1 mm) e inyectar suavemente AAC (6 &#39;)-Ii solución en la celda hasta que el exceso de líquido es visible por encima de la parte superior de la celda de muestra. </li><li> Lentamente levante la aguja aproximadamente 1 cm garantizando al mismo tiempo permanece en estado líquido en el desbordamiento. Retirar rápidamente y se inyecta una pequeña cantidad de solución (~ 0,25 ml) para eliminar las burbujas atrapadas en la celda de muestra. </li><li> Eliminar todos los desbordamiento de solución. Esto se logra haciendo deslizar suavemente la aguja al lado de desbordamiento en la celda de muestra. La punta de la jeringa se golpeó un saliente, éste es el deseo de la altura de la solución en ejecución. Eliminar todo el líquido que se encuentra encima de esta línea. </li></ol> 5. Cargando jeringa de inyección y el inicio de ejecución 14 <ol><li> Conecte la jeringa del tubo de carga de plástico en el puerto de llenado. </li><li> Émbolo menor de punta a la parte superior del puerto de llenado. </li><li> Ponga la solución en el tubo de AcCoA pipeta de llenado en la parte inferior del titular de la pipeta. La punta de la jeringa no debe tocar el fondo del tubo de llenado. </li><li> Dibujar lentamente la solución en la jeringa hasta que una pequeña cantidad entra en el tubo de la jeringa de carga. </li><li> Cerca del puerto de llenado mediante la reducción de la punta del émbolo y haga clic en el botón Cerrar puerto de llenado. Purgar y rellenar, haciendo clic en la Purga-&gt; botón de recarga, 3 veces para eliminar cualquier burbuja de aire que puedan haberse atrapado durante la carga. </li><li> Retire el tubo de llenado del titular de la pipeta y frote suavemente la punta de la jeringa. </li><li> Tome la Asamblea pipeta y jeringa suavemente más bajo en la celda de muestra. Proceder lentamente a medida que la jeringa se puede doblar fácilmente, por lo que se necesita gran cuidado. Asegúrese de que la jeringa se inserta por completo, presionando en la base del anillo de fijación. </li><li> Ajuste la temperatura deseada en ejecución (en este caso 20 ° C), y seleccione una potencia de referencia que es ligeramente mayor que la de inyección de flujo de calor máximo que se prevé (en este caso 20μcal/sec). </li><li> Programa de los volúmenes de inyección deseada y retrasos. (En este caso, 28 inyecciones fueron empleados. La primera inyección con un volumen de 2 l con un retraso de 60 s. Todas las inyecciones posteriores tuvieron un volumen de 10 l con retrasos en 330 s). </li></ol> 6. Ejecuta posterior <ol><li> En todas las ejecuciones posteriores, repita los pasos 2-5, mientras que la disminución AAC (6 &#39;)-II y las concentraciones de AcCoA en un factor de 2,4,8,16, y 32. </li></ol> 7. Análisis de Datos <ol><li> Utilizar un procedimiento de ajuste que a nivel mundial para todos de las isotermas de un único conjunto de parámetros de enlace, como se describió previamente 10. </li></ol> 8. Resultados representante Los datos representativos se muestran en la Figura 1. Las formas de las isotermas debe variar con la concentración. Más nítidas las transiciones que se espera para un mayor c-valores (es decir, la proteína más alta y las concentraciones de ligando) (Figura 2). En el caso de la AAC (6 &#39;)-II, el modelo secuencial de dos sitios le da un mejor ajuste de una descripción de dos conjuntos de sitios idénticos e independientes con estequiometría ajustable. <img src="/files/ftp_upload/2529/2529fig1.jpg" alt="Figura 1" /> Figura 1. Isotermas producido por la titulación de AcCoA (3,86 mm) en AAC (6 &#39;)-II (192 M). A) Raw rastro ITC. B) Los valores integrados para la determinación de parámetros de enlace (cuadrados) con un ajuste de 2 sitio secuencial (-). <img src="/files/ftp_upload/2529/2529fig2.jpg" alt="Figura 2" /> Figura 2. Isotermas de ITC para AcCoA titulada en AAC (6 &#39;)-II en varias concentraciones. Los datos experimentales (círculos) se ajustaron a un modelo de 2 conjuntos de sitios independientes (magenta discontinua) y un modelo de 2 sitio secuencial (color azul). El modelo de 2 sitio sequentional da claramente un acuerdo mejor en general. Las concentraciones empleadas fueron A) 6 M, 0,25 mm, B) 12 m, 0,25 mm, C) 24 mM, 0,5 mM, D) 48 mM, 1,0 mM, E) 96 mM, 1,9 mM, y F) 196 M, 3,86 mM, para AAC (6 &#39;)-II y AcCoA respectivamente.

<ol>
	<li>Cliff, M. J., Ladbury, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+the+year+2002+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2002 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 16, 383-391 (2003).</li><li>Cliff, M. J., Gutierrez, A., Ladbury, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+the+year+2003+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2003 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 17, 513-523 (2004).</li><li>Ababou, A., Ladbury, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survey+of+the+year+2004:+literature+on+applications+of+isothermal+titration+calorimetry.">Survey of the year 2004: literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 19, 79-89 (2006).</li><li>Ababou, A., Ladbury, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survey+of+the+year+2005:+literature+on+applications+of+isothermal+titration+calorimetry.">Survey of the year 2005: literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 20, 4-14 (2007).</li><li>Okhrimenko, O. k. s. a. n. a., J, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+the+year+2006+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2006 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 21, 1-19 (2008).</li><li>Bjelic, S., Jelesarov, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+the+year+2007+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2007 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 21, 289-312 (2008).</li><li>Leavitt, S., Freire, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+protein+binding+energetics+by+isothermal+titration+calorimetry.">Direct measurement of protein binding energetics by isothermal titration calorimetry.</a> Current Opinion in Structural Biology. 11, 560-566 (2001).</li><li>Wiseman, T., Williston, S., Brandts, J. F., Lin, L. -. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+measurement+of+binding+constants+and+heats+of+binding+using+a+new+titration+calorimeter.">Rapid measurement of binding constants and heats of binding using a new titration calorimeter.</a> Analytical Biochemistry. 179, 131-137 (1989).</li><li>Capaldi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+X-Ray+Structure+of+Zebrafish+(Danio+rerio)+Ileal+Bile+Acid-Binding+Protein+Reveals+the+Presence+of+Binding+Sites+on+the+Surface+of+the+Protein+Molecule.">The X-Ray Structure of Zebrafish (Danio rerio) Ileal Bile Acid-Binding Protein Reveals the Presence of Binding Sites on the Surface of the Protein Molecule.</a> Journal of Molecular Biology. 385, 99-116 (2009).</li><li>Freiburger, L. A., Auclair, K., Mittermaier, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidating+Protein+Binding+Mechanisms+by+Variable-c+ITC.">Elucidating Protein Binding Mechanisms by Variable-c ITC.</a> ChemBioChem. 10, 2871-2873 (2009).</li><li>Wybenga-Groot, L. E., Draker, K. -. a., Wright, G. D., Berghuis, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+an+aminoglycoside+6'-N-acetyltransferase:+defining+the+GCN5-related+N-acetyltransferase+superfamily+fold.">Crystal structure of an aminoglycoside 6'-N-acetyltransferase: defining the GCN5-related N-acetyltransferase superfamily fold.</a> Structure. 7, 497-507 (1999).</li><li>Draker, K., Northrop, D. B., Wright, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+Mechanism+of+the+GCN5-Related+Chromosomal+Aminoglycoside+Acetyltransferase+AAC(6')-Ii+from+Enterococcus+faecium:+Evidence+of+Dimer+Subunit+Cooperativity.">Kinetic Mechanism of the GCN5-Related Chromosomal Aminoglycoside Acetyltransferase AAC(6')-Ii from Enterococcus faecium: Evidence of Dimer Subunit Cooperativity.</a> Biochemistry. 42, 6565-6574 (2003).</li><li>Wright, G. D., Ladak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+and+characterization+of+the+chromosomal+aminoglycoside+6'-N-acetyltransferase+from+Enterococcus+faecium.">Overexpression and characterization of the chromosomal aminoglycoside 6'-N-acetyltransferase from Enterococcus faecium.</a> Antimicrob. Agents Chemother. 41, 956-960 (1997).</li><li>MicroCal. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ITC Data Analysis in Origin. , (2004).</li></ol>

Esta porción de análisis de la variable de ajuste-c se ha descrito anteriormente en detalle 10. Aquí mostramos los aspectos prácticos de la recolección variable c conjuntos de datos adecuados para este enfoque. Es esencial que todas las proteínas y las muestras de ligando se han extraído de las soluciones madre misma. Por lo tanto, es importante que la solución de un stock suficiente se preparó inicialmente para completar toda la serie de experimentos. Esto asegura que la proporción de AAC (6 &#39;)-II y AcCoA es constante entre todos los experimentos, y reduce las fluctuaciones aleatorias en la concentración en base a una muestra a muestra. En este caso, mucho cuidado se debe tomar en el manejo del ligando. AcCoA es químicamente inestable en solución por lo que es esencial que la solución madre permanece en estado líquido durante períodos muy cortos de tiempo. Una vez que la cantidad adecuada es alícuotas de la solución madre, la acción inmediata debe volver a congelar. Si esto no se hace, entonces el AcCoA puede degradarse con el tiempo y la concentración no será correcta en carreras posteriores. Una vez alícuotas, las muestras de AcCoA debe ser utilizado inmediatamente. En el caso de las proteínas que son inestables, las precauciones similares será necesario. Este procedimiento se puede modificar fácilmente para estudiar otros sistemas complejos. 10 Las concentraciones utilizadas deben adaptarse a las constantes de unión del sistema específico. Una amplia gama de valores de c entre 1 y 1.000 8,10 se requiere de todo el conjunto de datos.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Acetyl coenzyme A (AcCoA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A2056</td>
<td>&nbsp;</td>
<tr>
<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>7365-45-9</td>
<td>&nbsp;</td>
<tr>
<td>ethylenediaminetetraacetic acid (EDTA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>431788</td>
<td>&nbsp;</td>
<tr>
<td>Spectra/Por 2 Dialysis Tubing</td>
<td>&nbsp;</td>
<td>Spectrum Labs</td>
<td>132678</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Syringe Filter (0.2 &mu;m)</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>281445-477</td>
<td>&nbsp;</td>
<tr>
<td>Cellulos Nitrate Membrane Filters (0.45 &mu;m)</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>7184-004</td>
<td>&nbsp;</td>
<tr>
<td>VP-ITC</td>
<td>&nbsp;</td>
<td>MicroCal</td>
<td>VP-ITC</td>
<td>Microcalorimeter used for measurements</td>
</tr>
<tr>
<td>ThermoVac</td>
<td>&nbsp;</td>
<td>MicroCal</td>
<td>USB Thermo Vac</td>
<td>Temperature Controlled Degassing Station</td>
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collecting variable-concentration isothermal titration calorimetry datasets in order to determine binding mechanisms

Calorimetría de titulación isotérmica (ITC) se utiliza comúnmente para determinar los parámetros termodinámicos asociados a la unión de un ligando a una macromolécula de acogida. ITC tiene algunas ventajas sobre los enfoques comunes espectroscópicas para el estudio de host / ligando interacciones. Por ejemplo, el calor liberado o absorbido cuando los dos componentes interactúan se mide directamente y no requiere ningún periodista exógenos. Así, la entalpía de unión y de la constante de asociación (Ka) se obtienen directamente de los datos de ITC, y se puede utilizar para calcular la contribución entrópica. Además, la forma de la isoterma es dependiente de la c-valor y el modelo mecanicista involucrados. El valor de c se define como c = n [P] ATR, donde [P] t es la concentración de proteínas, y n es el número de sitios de unión del ligando dentro del huésped. En muchos casos, múltiples sitios de unión de un ligando dado no son equivalentes y el CCI permite la caracterización de los parámetros termodinámicos de unión para cada sitio de unión individual. Sin embargo, esto requiere que el modelo de enlace correcto utilizar. Esta elección puede ser problemático si los diferentes modelos pueden ajustarse a los datos experimentales. Hemos demostrado que este problema puede evitarse mediante la realización de experimentos en varias c-valores. Las isotermas de múltiples obtenidos en los diferentes valores de c-se ajustan al mismo tiempo a los modelos por separado. El modelo correcto es el siguiente identificados en base a la bondad de ajuste en toda la variable c conjunto de datos. Este proceso se aplica aquí a los aminoglucósidos resistencia a aminoglucósidos causan la enzima N-acetil-6&#39;-II (AAC (6 &#39;)-II). A pesar de nuestra metodología es aplicable a cualquier sistema, la necesidad de esta estrategia se demuestra mejor con un sistema de macromolécula-ligando muestra allostery o cooperatividad, y cuando los diferentes modelos de unión proporciona se ajusta esencialmente idénticos a los mismos datos. Por lo que sabemos, no hay sistemas disponibles en el mercado. AAC (6 &#39;)-II, es un homo-dímero con dos sitios activos, que muestra cooperatividad entre las dos subunidades. Sin embargo, los datos de ITC obtiene en un solo valor de c se puede estar en forma igual de bien que al menos dos modelos diferentes de un modelo de dos conjuntos de sitios independientes y de un modelo (cooperativa) de dos sitios secuencial. A través de la variación de la c-valor como se explicó anteriormente, se estableció que el modelo correcto de unión para AAC (6 &#39;)-II es un modelo de dos sitios de unión secuencial. En este documento, se describen los pasos que se deben tomar cuando se realizan experimentos de ITC para obtener datos adecuados para la variable c-análisis.

Watch this Scientific Journal Video about Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms at JoVE.com

Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms

ITC es una herramienta poderosa para el estudio de la unión de un ligando a su anfitrión. En los sistemas complejos sin embargo, varios modelos pueden adaptarse bien a los datos. El método descrito aquí proporciona un medio para dilucidar el modelo de encuadernación adecuado para los sistemas complejos y extraer los parámetros termodinámicos correspondientes.

Recogida de concentración variable conjuntos de datos calorimetría de titulación isotérmica para determinar los mecanismos de enlace

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host ...

collecting-variable-concentration-isothermal-titration-calorimetry

Nanyang Technological University

Research

JoVE Journal

Biology

12.7K vistas.  McGill University. ITC es una herramienta poderosa para el estudio de la unión de un ligando a su anfitrión. En los sistemas complejos sin embargo, varios modelos pueden adaptarse bien a los datos. El método descrito aquí proporciona un medio para dilucidar el modelo de encuadernación adecuado para los sistemas complejos y extraer los parámetros termodinámicos correspondientes.

Video: Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms

Investigating the Immunological Mechanisms Underlying Organ Transplant Rejection

La investigación de los mecanismos inmunológicos subyacentes rechazo de trasplante de órganos

Investigación

Biología

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds 

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. Collecting site-specific biochemical markers allows us to evaluate the relationship between surgical wound and serum levels; determine any associations between mediator release, pain and analgesic consumption; and evaluate the effect of systemic and peripheral drug administration on surgical wound biochemistry.
This methodology has been applied to healthy women undergoing elective cesarean delivery with spinal anesthesia. Wound exudate and serum mediators, in conjunction with pain scores and analgesics consumption were measured at 1, 6, 24, and 48 hours post-cesarean delivery. Biochemical mediators that were detected included IL-1&beta;, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNF&alpha;, INF&gamma;, G-CSF, GM-CSF, MCP-1 and MIP-1&beta;, nerve growth factor (NGF), prostaglandin E2 (PG-E2) and substance P. We found no correlations between wound and serum cytokines concentrations or time-release profiles (J Pain. 2008 Jul 9(7):650-7). This article describes and demonstrates the feasibility of collecting and assaying nociceptive and inflammatory mediators in surgical wounds at specific time points. The lack of significant correlations between serum and wound levels shows the importance of determining site-specific release if surgical wounds and localized pathologies are to be studied.

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds

A technique to collect and measure surgical wound biochemical mediators at specific time points.

La recogida y medición de mediadores inflamatorios y nociceptivos en las heridas quirúrgicas

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. ...

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
The described kit procedure detects the trypsin protease control at a concentration of approximately 0.5 &mu;g/ml (5 ng of trypsin added to the assay). This sensitivity can be increased with a longer incubation time, up to 24 hours. The assay is performed in microcentrifuge tubes and procedures are provided for fluorescence detection using either cuvettes or multiwell plates.

The Protease Fluorescent Detection Kit is designed for the measurement of protease activity using fluorometry. It is also suitable for detection of trace amounts of protease contamination. The method is based on the proteolytic hydroysis of a proprietary formulation of a FITC-labeled casein substrate.

El uso del kit de la proteasa detección fluorescente para determinar la actividad de la proteasa

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR-Clip - un método para identificar transcriptoma en todo los sitios de unión de las proteínas de unión a ARN

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

Cultura de embriones de ratón de pulmón, un sistema para evaluar los mecanismos moleculares de la ramificación

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given.

A general protocol for the use of isothermal titration calorimetry to monitor the binding thermodynamics for biological systems with moderate binding affinities is presented.

Calorimetría de titulación isotérmica para la medición de macromoléculas-ligando de afinidad

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction.  In biological ...

Culturing Primary Rat Inner Medullary Collecting Duct Cells

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP binds to vasopressin V2 receptors (V2R) on the surface of the cells and thereby induces synthesis of cAMP. This stimulates cellular signaling processes leading to changes in the phosphorylation of the water channel aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 and thereby triggers the translocation of AQP2 from intracellular vesicles into the plasma membrane facilitating water reabsorption from primary urine. Aberrations of AVP release from the pituitary or AVP-activated signaling in principal cells can cause central or nephrogenic diabetes insipidus, respectively; an elevated blood plasma AVP level is associated with cardiovascular diseases such as chronic heart failure and the syndrome of inappropriate antidiuretic hormone secretion.
Here, we present a protocol for cultivation of primary rat inner medullary collecting duct (IMCD) cells, which express V2R and AQP2 endogenously. The cells are suitable for elucidating molecular mechanisms underlying the control of AQP2 and thus to discover novel drug targets for the treatment of diseases associated with dysregulation of AVP-mediated water reabsorption. IMCD cells are obtained from rat renal inner medullae and are used for experiments six to eight days after seeding. IMCD cells can be cultured in regular cell culture dishes, flasks and micro-titer plates of different formats, the procedure only requires a few hours, and is appropriate for standard cell culture laboratories.

Arginine-vasopressin (AVP) controls fine-tuning of body water homeostasis through facilitating water reabsorption by renal principal cells. Here, we present a protocol for the cultivation of primary rat inner medullary collecting duct cells suitable for the elucidation of molecular mechanisms underlying AVP-mediated water reabsorption.

El cultivo primarios de rata medular interna recolección de células del conducto

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP ...

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

Titulación bioquímica de glucógeno<em&gt; In vitro</em

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized near the mitochondria. Support for this possibility is derived from recent studies, in which many mRNAs encoding mitochondrial proteins were shown to be localized to the mitochondria vicinity. Together with earlier demonstrations of ribosomes&#8217; association with the outer membrane, these results suggest a localized translation process. Such localized translation may improve import efficiency, provide unique regulation sites and minimize cases of ectopic expression. Diverse methods have been used to characterize the factors and elements that mediate localized translation. Standard among these is subcellular fractionation by differential centrifugation. This protocol has the advantage of isolation of mRNAs, ribosomes and proteins in a single procedure. These can then be characterized by various molecular and biochemical methods. Furthermore, transcriptomics and proteomics methods can be applied to the resulting material, thereby allow genome-wide insights. The utilization of yeast as a model organism for such studies has the advantages of speed, costs and simplicity. Furthermore, the advanced genetic tools and available deletion strains facilitate verification of candidate factors.

Many mRNAs encoding mitochondrial proteins are associated with the mitochondria outer membrane. We describe a subcellular fractionation procedure aimed at isolation of yeast mitochondria with its associated mRNAs and ribosomes. This protocol can be applied to cells grown under diverse conditions&#160;in order to reveal mechanisms of mRNA localization and localized translation near the mitochondria.

Aislamiento de ARNm asociados a la mitocondria de levadura para estudiar los mecanismos de traducción localizada

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Medición y Análisis de la producción de ácido extracelular para Determinar glicolítica Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...