Estamos muy agradecidos con el Dr. Thierry Pourcher para permitirnos usar el espectrómetro de fluorescencia y por su ayuda. El laboratorio está financiado por la Ligue Nationale Contre le Cancer (labellisée équipe), la Association pour la Recherche contre le Cáncer, el Instituto Nacional del Cáncer du (INCA), la Agence Nationale pour la Recherche, METOXIA (programa de la UE FP7), el Centro A. Lacassagne, el Centre National de la Recherche Scientifique, el Institut National de la Santé et de la Recherche Médicale y la Universidad de Niza ( <a href="http://www.unice.fr/isdbc/" target="_blank">http://www.unice.fr/isdbc/</a> ). Damos las gracias a la Dra. M Christiane Brahimi-Horn para la lectura crítica y la corrección editorial.

Titulación bioquímica de glucógeno 1. La lisis celular <ol><li> Se siembran las células a una concentración de 0,5 a 2 x 10 6 por placa de 100 mm de diámetro. </li><li> Tratamiento: incubar las células 24, 48, o 72 horas en baja concentración de oxígeno (hipoxia) en una estación de trabajo anaeróbico Bug-Box (Ruskinn Tecnología Biotrace International Plc, Bridgend, Reino Unido), fijado en el 1% o el 0,1% de O 2, 94% o 94,9% de N 2, y 5% de CO 2. En paralelo, se incuban las células en normoxia (21% de O 2, 5% de CO 2). Cambio de medio (medio que contiene glucosa-25 mM) cada 24 h para minimizar las variaciones en la concentración de glucosa durante el tiempo de experimento. </li><li> Después del tratamiento, las células de lavado 2 veces con PBS para eliminar las trazas de glucosa a partir de medio de cultivo. Raspar las células en PBS frío. </li><li> Centrifugar la solución a 1.000 rpm durante 5 min, descartar el sobrenadante y lavar el sedimento una vez con PBS. El pellet puede congelarse a -20 ° C antes de continuar.</li><li> Resuspender el sedimento en 100 l de agua destilada. Añadir 100 l de la hidrólisis del glucógeno Buffer se proporcionan con el kit de ensayo de glucógeno (Abcam). Hervir el lisado a 95 ° C durante 5 minutos para inactivar las enzimas. </li><li> Vórtice y se centrifuga el lisado a 13.000 rpm durante 10 min para eliminar los productos insolubles. Mantenga el sobrenadante. </li><li> Para normalizar los niveles de glucógeno para el contenido de proteínas entre muestras, la cuantificación de proteínas se puede hacer con 25-35 l del sobrenadante usando el ensayo del ácido bicinconínico (BCA-Interchim). </li></ol> Algunos de la lisado se puede utilizar para medir la concentración de glucosa libre en el interior de las células. 2. La hidrólisis del glucógeno <ol><li> Mezclar 50 l del sobrenadante (etapa 1) con 1 l de la hidrólisis enzimática de la mezcla. Esta mezcla contiene glucoamilasa. Incubar durante 30 min a temperatura ambiente. </li><li> Preparar la curva de calibración por dilución de la glucógeno estándar (2 mg / ml) supplied con el kit a una solución de 10 g / ml en el tampón de hidrólisis. A continuación, preparar seis tubos separados con 0, 4, 8, 12, 16, y 20 l de 10 mg estándar / ml y ajustar cada tubo a un volumen final de 50 l con tampón de hidrólisis para dar una concentración de glucógeno de 0, 0,04 , 0,08, 0,12, 0,16, y 0,2 g / ml. Añadir 1 l de hidrólisis enzimática de la mezcla en los estándares y se incuba durante 30 min a temperatura ambiente. </li></ol> El fondo de la concentración de glucosa de la línea celular, dada por el valor de la concentración de glucosa libre, se debe restar del valor de la concentración total de glucosa (glucosa a partir de la hidrólisis del glucógeno + sin glucosa) para determinar el nivel de glucógeno en la célula. 3. Desarrollo y lectura de fluorescencia <ol><li> Para cada condición, (tubo 1) preparar un tubo para medir la concentración de glucosa libre y dos tubos (tubos 2 y 3) para medir la concentración total de glucosa (cada ingenio tuboja diferente volumen de glucógeno hidrolizada). </li><li> Añadir 15 l de sobrenadante extraídas al final de la etapa 1 en el tubo 1. Añadir 35 l de tampón de hidrólisis para completar hasta un volumen final de 50 l. Fluorescencia obtenida dará la concentración de glucosa libre. </li><li> Añadir X l de glucógeno hidrolizado (obtenido en el paso 2) en el tubo 2. Ajustar a un volumen final de 50 l. Volumen de la muestra (X l) tiene que ser ajustado de acuerdo con la densidad celular y / o tipo de célula con el fin de obtener los valores de fluorescencia que no están más allá de las concentraciones de la curva de calibración. </li><li> Añadir 2 X l de glucógeno hidrolizada en el tubo 3. Ajustar a un volumen final de 50 l. </li><li> Preparar una solución de desarrollo para las muestras y los estándares mediante la mezcla para cada tubo 48,7 l de Buffer Desarrollo, 1 l de Enzyme Development Mix y 0,3 l de OxiRed Probe (suministrado en el kit de ensayo de glucógeno). Por ejemplo, para 2 condiciones, preparar una mezcla de 12 tubos (6 de las normas, 2 de glucosa libre y 4 para la fluorescencia total de glucosa). </li><li> Añadir 50 l de esta mezcla a cada tubo y se incuba durante 30 min a temperatura ambiente en la oscuridad. </li><li> Medir la fluorescencia a una longitud de onda de excitación de 535 nm, una longitud de onda de emisión de 587 nm como se recomienda, y una ranura de 3 nm para excitación y emisión. </li></ol>

<ol>
	<li>Pelletier, 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=Glycogen+Synthesis+is+Induced+in+Hypoxia+by+the+Hypoxia-Inducible+Factor+and+Promotes+Cancer+Cell+Survival.">Glycogen Synthesis is Induced in Hypoxia by the Hypoxia-Inducible Factor and Promotes Cancer Cell Survival.</a> Front Oncol. 2, (2012).</li><li>Alonso, M. D., Lomako, J., Lomako, W. M., Whelan, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+look+at+the+biogenesis+of+glycogen.">A new look at the biogenesis of glycogen.</a> FASEB J. 9, 1126-1137 (1995).</li><li>Bollen, M., Keppens, S., Stalmans, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+features+of+glycogen+metabolism+in+the+liver.">Specific features of glycogen metabolism in the liver.</a> Biochem. J. 336 (Pt 1), 19-31 (1998).</li><li>Parker, G. J., Koay, A., Gilbert-Wilson, R., Waddington, L. J., Stapleton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMP-activated+protein+kinase+does+not+associate+with+glycogen+alpha-particles+from+rat+liver.">AMP-activated protein kinase does not associate with glycogen alpha-particles from rat liver.</a> Biochem. Biophys. Res. Commun. 362, 811-815 (2007).</li><li>Sullivan, M. 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=Nature+of+alpha+and+beta+particles+in+glycogen+using+molecular+size+distributions.">Nature of alpha and beta particles in glycogen using molecular size distributions.</a> Biomacromolecules. 11, 1094-1100 (2010).</li><li>Chee, N. P., Geddes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+liver+glycogen.">The structure of liver glycogen.</a> FEBS Lett. 73, 164-166 (1977).</li><li>Roach, P. J., Depaoli-Roach, A. A., Hurley, T. D., Tagliabracci, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+and+its+metabolism:+some+new+developments+and+old+themes.">Glycogen and its metabolism: some new developments and old themes.</a> Biochem. J. 441, 763-787 (2012).</li><li>Moses, S. W., Bashan, N., Gutman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+metabolism+in+the+normal+red+blood+cell.">Glycogen metabolism in the normal red blood cell.</a> Blood. 40, 836-843 (1972).</li><li>Agbanyo, M., Taylor, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+3-deoxy-3-fluoro-D-glucose+into+glycogen+and+trehalose+in+fat+body+and+flight+muscle+in+Locusta+migratoria.">Incorporation of 3-deoxy-3-fluoro-D-glucose into glycogen and trehalose in fat body and flight muscle in Locusta migratoria.</a> Biosci. Rep. 6, 309-316 (1986).</li><li>Louzao, M. C., 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="Fluorescent+glycogen"+formation+with+sensibility+for+in+vivo+and+in+vitro+detection.">"Fluorescent glycogen" formation with sensibility for in vivo and in vitro detection.</a> Glycoconj. J. 25, 503-510 (2008).</li><li>Sheehan, D. C. H., B, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Theory and practice of histotechnology. , (1980).</li><li>Baba, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+monoclonal+antibody+that+recognizes+glycogen+and+its+application+for+immunohistochemistry.">Production of monoclonal antibody that recognizes glycogen and its application for immunohistochemistry.</a> Kokubyo Gakkai Zasshi. 60, 264-287 (1993).</li></ol>

Los autores declaran no tener ningún conflicto de intereses.

Valoración bioquímica de la glucógeno in vitro permite una cuantificación precisa de las células el contenido de glucógeno. En comparación con algunas otras técnicas (PAS, de inmunofluorescencia con un anticuerpo glucógeno, etc.), Esta valoración es muy específico, sensible y reproducible. Por otra parte, el método es conveniente ya que no requiere la radiactividad pero un espectrómetro de fluorescencia. Sin embargo, esta técnica es puramente cuantitativa y no proporciona información sob...

El glucógeno es un polímero multibranched de residuos de glucosa, que está presente en el citoplasma de muchos tipos de células. Es una de las principales formas de almacenamiento de energía en las células y juega un papel importante en el metabolismo de la glucosa. La mayoría de las células de mamífero son capaces de producir y glucógeno tienda, que puede ser degradado rápidamente en glucosa para promover la glicolisis y la producción de ATP durante el estrés metabólico. Los hepatocitos producen cantidades masivas de glucógeno para regular el nivel de azúcar en la sangre proporcionando con ello un suministro continuo de glucosa para el cuerpo. En contraste, la concentración de glucógeno en otras células (músculos, células rojas de la sangre, etc) es relativamente bajo. Sin embargo, a nivel local, estas cantidades son suficientes para proporcionar energía a corto plazo, cuando las células están expuestas súbitamente a un entorno privado de nutrientes. La síntesis de glucógeno y la degradación del glucógeno sigue los mismos pasos en todos los tejidos (Figura 1). En primer lugar, la glucosaentra en las células por transportadores de glucosa (GLUT), y se convierte rápidamente a partir de glucosa-6-fosfato (G6P) en glucosa-1-fosfato (G1P) por fosfoglucomutasa. G1P se modifica luego en UDP-glucosa, y el carbono C1 de UDP-glucosa se une a un residuo de tirosina de glucogenina, la proteína de anclaje de glucógeno. Esta molécula, considerado un cebador de glucógeno, se extiende por la unión de la UDP-glucosa a la glucosa terminal por un α (1 → 4) unión a través de la glucógeno sintasa. Finalmente, cuando se forma una cadena lineal de más de 11 residuos de glucosa, la enzima de ramificación transfiere un oligosacárido terminal de constituido por un mínimo de 6 residuos de glucosa a otro de glucosa de la cadena cerca a través de una α (1 → 6) de ligamiento. La repetición de este proceso da una estructura fractal masiva que contiene ramas que forman una hélice con 6,5 glucosa por turno. El glucógeno puede ser inversamente hidrolizada a glucosa por la acción concertadadesramificante de enzimas que hidrolizan la α (1 → 6) unido y la glucógeno fosforilasa que hidroliza la α (1 → 4) glicosídico límite entre el último residuo de glucosa de una rama y la molécula de glucógeno. Esta reacción llamada glucogenolisis se activa mediante el aumento de los niveles de AMP (que refleja el consumo de ATP), y se inhibe por la glucosa y ATP 2,3. Por microscopía electrónica, las moléculas de glucógeno se han descrito en muchos tipos de células como partículas β libres (o monopartículas glucógeno) de 15-30 nm de diámetro. En determinados tipos de células tales como hepatocitos, partículas β pueden ser ensamblados en un complejo para formar rosetas, también conocidos como partículas α que varían en diámetro desde 80 nm a un máximo de 200 nm 4 </sup&gt;. La forma en que estas partículas β se unen para formar grupos más grandes de partículas α todavía no está completamente aclarada. Alguna evidencia tiende a demostrar que las partículas β pueden estar unidos por enlace covalente 5, enlace de hidrógeno, o incluso a través de interacciones proteína-proteína 6. La cantidad de glucógeno almacenado en las células depende de muchos parámetros: (I) la cantidad de glucogenina en la célula que inicia la síntesis de glucógeno, (ii) la actividad de la sintasa de glucógeno fosforilasa y regulada por la fosforilación de proteínas / desfosforilación; (III) la concentración de la glucosa en las células, que depende de varios parámetros, tales como el suministro de glucosa a partir del sistema vascular y la captación de glucosa por las células. Las reservas de glucógeno son estrictamente regulados por la regulación alostérica de hormonas biosintéticas través de metabolitos intermedios, por las hormonas que regulan el metabolismo de la energía, y por vías de señalización de detección de nutrientes 7. Es importante estarcapaces de cuantificar glucógeno en muestras biológicas para entender mejor la importancia del metabolismo del glucógeno en todo el cuerpo y nivel celular. Se describe aquí un bioquímica precisa, reproducible y conveniente en el ensayo in vitro de glucógeno. Esta técnica se basa en la fluorescencia de la glucosa cuantificación antes y después de la hidrólisis específica de glucógeno. Existen otros métodos para estimar el nivel de glucógeno en las células, pero la mayoría de ellos no son cuantitativos. Una de las primeras técnicas descritas para la cuantificación de glucógeno en las células se basó en la medición de la incorporación de [14 C]-glucosa en glucógeno 8,9. El uso de radiactividad que hace que este proceso sea más difícil de manejar, pero tiene la ventaja de proporcionar la tasa de incorporación de glucosa en glucógeno y en la distinción entre la distribución de los residuos de glucosa en las ramas exteriores y en el núcleo de la molécula (que también requiere una β-amyloly adicionalsis y un paso cromatográfico). Otra técnica fue desarrollada más recientemente y se basa en la incorporación de 2-NBDG (2 - {N-[7-nitrobenz-2-oxa-1 ,3-diazol-4-il] amino}-2-desoxiglucosa), una derivado fluorescente 2-desoxiglucosa, en glucógeno 10. La intensidad de fluorescencia medida refleja la cantidad de glucógeno producido y se puede medir con un lector de fluorescencia. Distribución de la fluorescencia en la célula también puede ser evaluada por microscopía confocal. Entre las otras técnicas no cuantitativas, la tinción de ácido periódico de Schiff (PAS) es quizás el más común. Se puede utilizar para la detección de glucógeno en células fijadas, secciones de tejido en parafina o congelado. Este histológicos colores técnica no específicamente polisacáridos, glicolípidos, glicoproteínas, celulosa y mucinas neutras, de color morado. La especificidad de esta prueba se puede aumentar mediante el tratamiento de células o secciones de tejido fijadas con diastasa, que digiere específicamente glucógeno. A partir de entonces,el nivel de glucógeno puede estimarse cualitativamente comparando muestras no hidrolizadas (todos macromoléculas carbohidrato modificado) a las muestras hidrolizadas (carbohidratos macromoléculas modificado excepto glucógeno). La tinción de PAS y el análisis microscópico, a diferencia de los ensayos bioquímicos de glucógeno, proporciona información relativa a la distribución de glucógeno en la célula, que puede ser difusa o concentrada en una cierta parte de la célula. Sin embargo, a pesar de que las estimaciones de tinción PAS diferencias en la acumulación de glucógeno entre diferentes condiciones, no es cuantitativo 11. Un anticuerpo monoclonal de ratón originalmente hizo usando el cartílago condilar mandibular como el antígeno se ha demostrado que reaccionar con glucógeno en las células y con glucógeno purificada in vitro 12. Como este anticuerpo reconoce específicamente las cadenas de azúcar relacionadas glucógeno-, es una herramienta útil para la detección de glucógeno por inmunohistoquímica en una manera más específica que la tinción de PAS. </p&gt; La microscopía electrónica es otra técnica que permite la visualización de los granos de glucógeno en las células y la evaluación del grado de almacenamiento de glucógeno. De hecho, las partículas β de glucógeno son fácilmente reconocibles con una microscopía electrónica como gránulos electrón densos 1.

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			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>31966.047</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Glycogen Assay Kit</td>
			<td>Abcam</td>
			<td>ab65620 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>PBS</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		<tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
	 </tr>
		 <tr>
			<td>Material Name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments (optional)</td>
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		 <tr>
			<td>Fluorescence Spectrometer</td>
			<td>PerkinElmer</td>
			<td>LS 50B</td>
			<td></td>
		 </tr>
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 </table>

biochemical titration of glycogen in vitro

El glucógeno es la principal de polímero energético de la glucosa en animales vertebrados y juega un papel crucial en el metabolismo de todo el cuerpo, así como en el metabolismo celular. Ya existen muchos métodos para detectar glucógeno pero sólo unos pocos son cuantitativos. Se describe aquí un método que utiliza el kit de ensayo de Abcam glucógeno, que se basa en la degradación específica de glucógeno en glucosa por glucoamilasa. La glucosa se oxida específicamente a un producto que reacciona con la sonda OxiRed para producir fluorescencia. La titulación es preciso, sensible y se puede lograr en extractos de células o secciones de tejido. Sin embargo, en contraste con otras técnicas, no da información sobre la distribución de glucógeno en la célula. Como ejemplo de esta técnica, como se describe aquí la titulación de glucógeno en dos líneas celulares, chino CCL39 fibroblastos de pulmón de hámster y LS174 carcinoma de colon humano, se incubaron en normoxia (21% de O 2) frente a la hipoxia (1% de O 2). La hipótesis de que la hipoxia es un signal que prepara las células para sintetizar y almacenar glucógeno para sobrevivir 1.

Un nivel bajo de oxígeno (hipoxia) en señales de tumores a las células tumorales la necesidad de almacenar energía para manejar agotamiento de los nutrientes posterior, a fin de sobrevivir. Como glucógeno es la principal polímero energético de la glucosa en las células de mamíferos, se estudió la regulación del almacenamiento de glucógeno en la hipoxia. El cálculo y la normalización de la concentración de glucógeno en los lisados ​​celulares se deben realizar en los datos en bruto de la fluorescencia...

Watch this Scientific Journal Video about Biochemical Titration of Glycogen In vitro at JoVE.com

Biochemical Titration of Glycogen In vitro

Se describe aquí un método bioquímico exacta, reproducible y conveniente para la titulación de glucógeno in vitro. Esta técnica utiliza el kit de ensayo de Abcam glucógeno y se basa en la hidrólisis sucesivas de glucógeno en glucosa y la glucosa titulación por fluorescencia.

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 ...

biochemical-titration-of-glycogen-in-vitro

Research

JoVE Journal

Biology

Biochemical Titration of Glycogen In vitro

28.2K vistas.  University of Nice - Sophia Antipolis.  Se describe aquí un método bioquímico exacta, reproducible y conveniente para la titulación de glucógeno in vitro. Esta técnica utiliza el kit de ensayo de Abcam glucógeno y se basa en la hidrólisis sucesivas de glucógeno en glucosa y la glucosa titulación por fluorescencia. 

Video: Biochemical Titration of Glycogen In vitro

Titration of Human Coronaviruses Using an Immunoperoxidase Assay

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for viruses that do not cause significant cytopathic effects, which is the case for prototype strains 229E and OC43 of human coronavirus (HCoV). Therefore, an alternative indirect immunoperoxidase assay (IPA) was developed for the detection and titration of these viruses and is described herein. Susceptible cells are inoculated with serial logarithmic dilutions of virus-containing samples in a 96-well plate format. After viral growth, viral detection by IPA yields the infectious virus titer, expressed as 'Tissue Culture Infectious Dose 50 percent' (TCID50). This represents the dilution of a virus-containing sample at which half of a series of laboratory wells contain infectious replicating virus. This technique provides a reliable method for the titration of HCoV-229E and HCoV-OC43 in biological samples such as cells, tissues and fluids. This article is based on work first reported in Methods in Molecular Biology (2008) volume 454, pages 93-102.

In this video, we demonstrate an alternative method for detection and titering of viruses using an enzymatic antigen detection technique known as an immunoperoxidase assay. Here, we will show you how to collect your viral samples, prepare the cells for testing, and finally the immunoperoxidase assay using serial dilutions to determine the viral titer.

La titulación de coronavirus humanos mediante un ensayo de inmunoperoxidasa

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for ...

Investigación

Biología

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

Cerebro del pez cebra ventrículo inyección

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

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

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host macromolecule. ITC has some advantages over common spectroscopic approaches for studying host/ligand interactions. For example, the heat released or absorbed when the two components interact is directly measured and does not require any exogenous reporters. Thus the binding enthalpy and the association constant (Ka) are directly obtained from ITC data, and can be used to compute the entropic contribution. Moreover, the shape of the isotherm is dependent on the c-value and the mechanistic model involved. The c-value is defined as c = n[P]tKa, where [P]t is the protein concentration, and n is the number of ligand binding sites within the host. In many cases, multiple binding sites for a given ligand are non-equivalent and ITC allows the characterization of the thermodynamic binding parameters for each individual binding site. This however requires that the correct binding model be used. This choice can be problematic if different models can fit the same experimental data. We have previously shown that this problem can be circumvented by performing experiments at several c-values. The multiple isotherms obtained at different c-values are fit simultaneously to separate models. The correct model is next identified based on the goodness of fit across the entire variable-c dataset. This process is applied here to the aminoglycoside resistance-causing enzyme aminoglycoside N-6'-acetyltransferase-Ii (AAC(6')-Ii). Although our methodology is applicable to any system, the necessity of this strategy is better demonstrated with a macromolecule-ligand system showing allostery or cooperativity, and when different binding models provide essentially identical fits to the same data. To our knowledge, there are no such systems commercially available. AAC(6')-Ii, is a homo-dimer containing two active sites, showing cooperativity between the two subunits. However ITC data obtained at a single c-value can be fit equally well to at least two different models a two-sets-of-sites independent model and a two-site sequential (cooperative) model. Through varying the c-value as explained above, it was established that the correct binding model for AAC(6')-Ii is a two-site sequential binding model. Herein, we describe the steps that must be taken when performing ITC experiments in order to obtain datasets suitable for variable-c analyses.

ITC is a powerful tool for studying the binding of a ligand to its host. In complex systems however, several models may fit the data equally well. The method described here provides a means to elucidate the appropriate binding model for complex systems and extract the corresponding thermodynamic parameters.

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 ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

Extracción de sangre para análisis bioquímicos en adultos de pez cebra

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

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 ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

Bioquímica y de Evaluación de Rendimiento microscópica de masa grasa en<em&gt; Caenorhabditis elegans</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

De Alto Rendimiento La titulación de luciferasa que expresan virus recombinantes

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

Los enfoques genéticos y bioquímicos para<em&gt; En Vivo</em&gt; y<em&gt; In Vitro</em&gt; Evaluación de la oligomerización de proteínas el receptor de rianodina Estudio de caso

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

Saccharomyces cerevisiae Exponential Growth Kinetics in Batch Culture to Analyze Respiratory and Fermentative Metabolism

Saccharomyces cerevisiae cells in the exponential phase sustain their growth by producing ATP through fermentation and/or mitochondrial respiration. The fermentable carbon concentration mainly governs how the yeast cells generate ATP; thus, the variation in fermentable carbohydrate levels drives the energetic metabolism of S. cerevisiae. This paper describes a high-throughput method based on exponential yeast growth to estimate the effects of concentration changes and nature of the carbon source on respiratory and fermentative metabolism. The growth of S. cerevisiae is measured in a microplate or shaken conical flask by determining the optical density (OD) at 600 nm. Then, a growth curve is built by plotting OD versus time, which allows identification and selection of the exponential phase, and is fitted with the exponential growth equation to obtain kinetic parameters. Low specific growth rates with higher doubling times generally represent a respiratory growth. Conversely, higher specific growth rates with lower doubling times indicate fermentative growth. Threshold values of doubling time and specific growth rate are estimated using well-known respiratory or fermentative conditions, such as non-fermentable carbon sources or higher concentrations of fermentable sugars. This is obtained for each specific strain. Finally, the calculated kinetic parameters are compared with the threshold values to establish whether the yeast shows fermentative and/or respiratory growth. The advantage of this method is its relative simplicity for understanding the effects of a substance/compound on fermentative or respiratory metabolism. It is important to highlight that growth is an intricate and complex biological process; therefore, preliminary data from this method must be corroborated by the quantification of oxygen consumption and accumulation of fermentation byproducts. Thereby, this technique can be used as a preliminary screening of compounds/substances that may disturb or enhance fermentative or respiratory metabolism.

Saccharomyces cerevisiae Exponential Growth Kinetics in Batch Culture to Analyze Respiratory and Fermentative Metabolism

Here we present a protocol to estimate the respiratory and fermentative metabolism by fitting the exponential growth of Saccharomyces cerevisiae to the exponential growth equation. Calculation of the kinetic parameters allows for the screening of influences of substances/compounds on fermentation or mitochondrial respiration.

Saccharomyces cerevisiae Cinética de crecimiento exponencial en la cultura de lote para analizar el metabolismo respiratorio y fermentativo

Saccharomyces cerevisiae cells in the exponential phase sustain their growth by producing ATP through fermentation and/or mitochondrial respiration. The ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

In vitro Análisis de la función E3 Ubiquitina Ligasa

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...