Este trabajo fue apoyado por el premio NSF # 0920274 y Carolina del Sur de la Estación Experimental del proyecto (SC-1700340) para KSS. Este documento es el N º 5929 Contribución Técnica de la Estación Experimental de la Universidad de Clemson.

El esquema general de este protocolo se describe en la figura 1. 1. Preparación para la solución de las curvas de calibración y ensayos <ol><li> Preparar 100 ml de una 2 mol / L de hidroxilamina-HCl. Pesar 13,9 g de clorhidrato de hidroxilamina (MW 69,49 g / mol) y se disuelven en aproximadamente 50 ml de agua destilada, agua desionizada (DDC 2 O). Ajustar el pH a 7.0 con pellets de hidróxido de potasio o una solución concentrada. Llevar el volumen final de 100 ml. La solución se puede almacenar a temperatura ambiente durante un máximo de 30 días o menos 4 ° C durante un máximo de 90 días. </li><li> Preparar 100 ml de cloruro férrico una / solución de ácido clorhídrico. Pesar 13,5 g de cloruro férrico (MW 270,32 g / mol) y se disuelven en aproximadamente 50 ml ddH 2 O. Añadir 41,3 ml de ácido clorhídrico concentrado (12,1 mol / L) y llevar a un volumen final de 100 ml. La concentración final de cloruro férrico será de 0,5 mol / L y la concentración final de ácido clorhídrico será de 5 mol/ L. La solución se almacena a temperatura ambiente. </li><li> Preparar 100 ml de una 1.84 mol / L de ácido trichoroacetic. Pesar 30,0 g de ácido tricloroacético (MW 163,39 g / mol), se disuelven en ddH2O y llevar el volumen final de 100 ml. La solución se almacena a temperatura ambiente. </li><li> Preparar 5 ml de un 0,1 mol / L solución de fosfato de acetilo. Pesar 0,092 g de fosfato de acetilo (MW 184,06 g / mol), se disuelven en ddH2O y llevar el volumen final de 5 ml. Alícuota de la solución a tubos de microcentrífuga (0,25 ml por tubo) y congelar a -20 ° C hasta su uso. Solución descongelada debe ser colocado en hielo y se puede utilizar durante aproximadamente 1 hora. Solución de fosfato de acetil que ha sido descongelado no debe volver a congelarse y reutilizarse. </li><li> Preparar 5 ml de un 0,1 mol / L de ADP. Pesar 0,24 g de ADP (MW 472,5 g / mol), se disuelven en ddH2O, y llevar el volumen final de 5 ml. Alícuota de la solución en tubos de microcentrífuga (0,5 ml por tubo) y congelar a -20 ° C hastauso. Mantener la solución descongelada en el hielo hasta su uso. </li><li> Prepare 50 ml de una 1 mol / L de Tris. Pesar 6,06 g de Tris (MW 121,14 g / mol), y se disuelven en 40 ml ddH 2 O. Ajustar el pH de la solución a 7,0 con ácido clorhídrico y alcanzar un volumen final de 50 ml. Guarde la solución a temperatura ambiente. </li><li> Prepare 10 ml de un 1 mol / L de solución de cloruro de magnesio. Pesar 2,03 g de cloruro de magnesio (MW 203,31 g / mol), se disuelven en ddH2O, y alcanzar un volumen final de 10 ml. Guarde la solución a temperatura ambiente. </li><li> Prepare 25 ml de solución de desarrollo mediante la mezcla de volúmenes iguales de la solución de ácido de cloruro férrico / clorhídrico y la solución de ácido tricloroacético. Solución de desarrollo deben estar preparados frescos cada día y se almacena a temperatura ambiente. </li></ol> 2. Preparación de una curva estándar de fosfato acetil <ol><li> Una curva estándar de fosfato de acetilo se genera utilizando cantidades conocidas de acetil fosfato. Una curva estándar de seis a eighpuntos t es adecuado. Las normas deben variar entre 0,03 a 0,9 moles moles por cada 300 l, que se correlaciona con la concentración final de 0,1 a 3 mmol / L. Diluir la solución de fosfato de valores acetilo a las concentraciones adecuadas de solución de Tris 100 mM en un volumen final de 300 mL. Una muestra de control sin acetil fosfato también deben estar preparados. </li><li> Incubar cada estándar y el control de los 37 ° C en un bloque de calor durante 1 minuto. </li><li> Añadir 50 l de la solución de clorhidrato de hidroxilamina a cada norma y el control y la mezcla agitando tres veces. Coloque el estándar en un bloque de 60 ° C de calor y se incuba durante cinco minutos. </li><li> Añadir 100 ml de solución de desarrollo de las normas y el control, la mezcla agitando tres veces, y permitir que se desarrolle el color de al menos un minuto a temperatura ambiente. </li><li> Centrífuga con todas las normas y el control en una microcentrífuga a 18.000 xg durante 1 minuto. </li><li> Mediciones de las muestras por espectrofotometría unt 540 nm con cubetas de plástico de 1 ml, utilizando el control como el espacio en blanco para espectrofotometría. </li><li> Generar una curva estándar utilizando el análisis de datos y el software adecuados de gráficos (por ejemplo, KaleidaGraph, Synergy Software). Valores de R 2 de 0.99 o superior son deseables. Los datos se representan como la absorbancia a 540 nm frente a moles de fosfato presente acetilo. </li></ol> 3. Ensayo para la actividad quinasa de acetato <ol><li> Utilizando las soluciones descritas, prepare una mezcla de reacción que contiene una concentración final de 0,1 buffer mol / L Tris, 10 mmol / L de cloruro de magnesio, 5 mmol / L de ADP, y 2 mmol / L acetil fosfato. Una mezcla de 10 ml de reacción se requieren 8,2 ml ddH2O, 1,0 ml de solución de Tris, 0,1 ml de solución de cloruro de magnesio, 0,5 ml de solución de ADP, y 0,2 ml de solución de fosfato de acetilo. Distribuir la mezcla de reacción de 300 alícuotas a tubos de microcentrífuga. Para la determinación de parámetros cinéticos y la optimización de las concentraciones de sustrato, un sustrato puede servariado y la otra sostenía a una concentración constante en las reacciones. </li><li> Pre-incubar la reacción durante 1 minuto a 37 ° C en un bloque de calor. </li><li> Agregar la cantidad deseada de enzima a la reacción y devolvió el tubo en el bloque de 37 º C de calor. También incluyen una reacción de control para que no se añade la enzima. Si las reacciones se realizan varias, añadir la enzima a los tubos a intervalos de tiempo específicos. Asegúrese de mantener la noción del tiempo. (NOTA: La cantidad de enzima que se utilizará tendrá que ser optimizado para asegurar que todos los de la acetil fosfato o sustrato ADP no se agotará - véase la sección de discusión) </li><li> Después de 5 minutos, agregar 50μl de la solución de clorhidrato de hidroxilamina a cada reacción y el control, mezclar y colocar los tubos en un bloque de 60 ° C de calor. Si varias reacciones se llevan a cabo, esto debe hacerse en el mismo intervalo de tiempo que la enzima se añadió. </li><li> Incubar las reacciones y controlar el durante 5 minutos a 60 ° C. </li><li> Añadir 100 ymu; L de la solución de desarrollo de cada reacción y el control, mezclar y colocar en un soporte de tubos en el banco de laboratorio. De formación de color por lo menos durante 1 minuto. </li><li> Centrifugar todos los tubos en una microcentrífuga a 18.000 xg durante 1 minuto. </li><li> Mediciones de muestras con un espectrofotómetro a 540 nm con cubetas de plástico de 1 ml y determinar la cantidad de fosfato presente acetil en comparación con la curva estándar de fosfato de acetilo. </li><li> La cantidad de sustrato acetil fosfato reducido se puede determinar restando la cantidad de acetil fosfato que queda después de la reacción enzimática de la cantidad de acetil fosfato presente en la enzima de control insuficiente. Realizar análisis de datos a partir de datos de gráficos y el software adecuados (por ejemplo, KaleidaGraph, Synergy Software). R 2 valores superiores a 0,97 son deseables. </li></ol> 4. Los resultados representativos: El propósito de este ensayo es medir la actividad de acetato quinasa en la directaión de la formación de acetato. Esto se hace mediante la medición de consumo de sustrato acetil fosfato, ya que no existe una medición sencilla y directa para la producción de acetato. Acetil fosfato que queda después de un momento dado durante la reacción enzimática se convierte en hidroxamato acetil y, posteriormente, al complejo hidroxamato férrico, que tiene un color rojizo (Figura 2) que se puede medir a 540 nm. La curva estándar de absorbancia frente trazado actual moles acetil fosfato en las normas se utiliza para determinar la cantidad de acetil fosfato restante en cada reacción. La curva estándar debe ser lineal a través del punto cero. Una curva estándar representativo se muestra en la Figura 3 tiene una pendiente de 1,2332 unidades de absorbancia por presentar mol acetil fosfato y un valor de R 2 de 0,99, indicando que los datos se ajusta a la ecuación lineal también. La ecuación para el ajuste lineal a los datos de la curva estándar se utiliza para el cálculo de la cantidad de acetil fosfato restante en la reacción. Los molesde acetil fosfato consumido se determina como la diferencia entre los moles de acetil fosfato presente en la reacción de control que carecen de la enzima y la moles de acetil fosfato que queda después de la reacción enzimática. Purificada, recombinante M. quinasa thermophila acetato fue ensayada mediante el protocolo descrito. Para el experimento se muestra en la Figura 4, la concentración de ADP se mantuvo constante en 5 mmol / L y la concentración de acetil fosfato en la reacción fue variada para determinar el m K de acetil fosfato. Como era de esperar, este experimento demuestra que la enzima siguiente estándar de Michaelis-Menten, como la cinética de cada sustrato y produce una curva de saturación hiperbólica al trazar la actividad frente a la concentración de fosfato de acetilo. La aparente K m valor de acetil fosfato fue de 0,27 ± 0,01 mmol / L, que se compara favorablemente con el valor publicado K m de 0,47 ± 0,01 mmol / L, junto con el dedecir 13. El ensayo directo también se puede utilizar para medir la actividad de las quinasas de acetato que utilizan otros sustratos de la ATP, que no es posible con la prueba estándar acoplados. Recombinante E. histolytica acetato quinasa 16, que produce PP i en lugar de ATP en la dirección de acetato de formación, fue sometido a esta prueba con fosfato de sodio en lugar de ADP. La concentración de fosfato de sodio en la reacción se mantuvo constante a 0,2 mol / L y la concentración de acetil fosfato era variada. En cuanto a la M. thermophila quinasa acetato, la E. quinasa histolytica acetato seguido de Michaelis-Menten, como la cinética de acetil fosfato y una curva de saturación hiperbólica se observó (Figura 5). La aparente K m valor de acetil fosfato fue de 0,50 ± 0,006 mmol / L. Reeves y Guthrie 15 determina un valor de K m de 0,06 mmol / L de acetil fosfato de la enzima nativa, cómo<html> embargo, la enzima fue parcialmente purificada y su ensayo involucró a cuatro enzimas de acoplamiento que también fueron sólo parcialmente purificada. Por lo tanto, es difícil comparar directamente estos valores. Protocolo de Régimen <ol><li> Generar una curva estándar de fosfato de acetilo </li><li> Diluir la enzima a la concentración deseada </li><li> Prepare la mezcla de reacción y la alícuota de 300 l con tubos de microcentrífuga </li><li> Reacciones preincubar a 37 ° C durante 1 minuto </li><li> Inicio reacciones mediante la adición de enzimas a intervalos de tiempo específicos </li><li> Añadir hidroxilamina en los mismos intervalos de detener las reacciones y se incuba a 60 ° C durante 5 minutos </li><li> Añadir cloruro férrico / solución de ácido tricloroacético para el desarrollo de color </li><li> Centrifugar los tubos a 18.000 xg durante 1 minuto </li><li> Medir la absorbancia a 540 nm </li><li> Determinar la cantidad de acetil fosfato consumido en comparación con la curva estándar </li></ol>tp_upload/3474/3474fig1.jpg &quot;/&gt; Figura 1. Esquema de Protocolo <img alt="Figura 2" src="/files/ftp_upload/3474/3474fig2.jpg" /> Figura 2. Normas acetil fosfato. Aumento de las concentraciones de fosfato de acetilo reacciona con hidroxilamina y el color se desarrolló tal como se describe. El control que carecen de acetil fosfato es de color amarillo brillante, y las reacciones que contienen cantidades crecientes de acetil fosfato son sucesivamente de color más oscuro. Este cambio de color se mide a 540 nm. <img alt="Figura 3" src="/files/ftp_upload/3474/3474fig3.jpg" /> Figura 3. Ejemplo de curva estándar acetil fosfato. Cantidades variables de acetil fosfato se hicieron reaccionar con hidroxilamina y el desarrollo de soluciones de color y se midió la absorbancia a 540 nm. La absorbancia a 540 nm frente a fosfato de moles acetil fue trazada y un ajuste lineal de los datos se aplicó. &lt;/ P&gt; <img alt="Figura 4" src="/files/ftp_upload/3474/3474fig4.jpg" /> Figura 4. Utilización de acetil fosfato por el M. ATP que producen thermophila quinasa acetato. La actividad enzimática se determinó en la dirección de la formación de acetato en presencia de 5 mmol / L de ADP con diferentes concentraciones de acetil fosfato. La cantidad consumida de acetil fosfato se determinó como la diferencia entre el importe de partida de acetil fosfato en la reacción y la cantidad de acetil fosfato que queda después de la reacción enzimática. Los ensayos se realizaron por triplicado con barras de error se muestra. <img alt="Figura 5" src="/files/ftp_upload/3474/3474fig5.jpg" /> Figura 5. Utilización de acetil fosfato por el PP i-producción de E. histolytica quinasa acetato. La actividad enzimática en la dirección de la formación de acetato se determinó en presencia de 0.2 mol / L de fosfato de sodio con diferentes concentraciones de acetil fosfato. La cantidad consumida de acetil fosfato se determinó como la diferencia entre el importe de partida de acetil fosfato en la reacción y la cantidad de acetil fosfato que queda después de la reacción enzimática. Los ensayos se realizaron por triplicado con barras de error se muestra.

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La detección de acetil fosfato en este ensayo depende de una suficiente concentración de clorhidrato de hidroxilamina y la concentración y la acidez de la solución de cloruro férrico. Alteración del volumen de ensayo requiere la reconsideración de estos dos componentes. Las reacciones enzimáticas que se describe aquí se realizaron a 37 ° C con la terminación hidroxilamina realiza a 60 ° C durante 5 minutos. Esta temperatura más alta es fundamental para permitir la rápida transformación de la acetil fosfat...

<table border="1"><tr><td width="160"> Nombre del reactivo </td><td width="108"> Empresa </td><td width="134"> Número de catálogo </td><td width="103"> Comentarios </td></tr><tr><td> Acetil fosfato </td><td> Sigma Aldrich </td><td> 01409 </td><td> La sal de litio (97%) </td></tr><tr><td> Fosfato de sodio monobásico (deshidratación) </td><td> ThermoFisher </td><td> S381 </td><td></td></tr><tr><td> Fosfato de sodio dibásico (anhidro) </td><td> ThermoFisher </td><td> S374 </td><td></td></tr><tr><td> Cloruro de magnesio (hexahidratado) </td><td> ThermoFisher </td><td> M33 </td><td></td></tr><tr><td> Tris Base </td><td> ThermoFisher </td><td> B152 </td><td></td></tr><tr><td> Cloruro férrico (hexahidratado) </td><td> ThermoFisher </td><td> I88 </td><td></td></tr><tr><td> El ácido tricloroacético </td><td> ThermoFisher </td><td> A324 </td><td></td></tr><tr><td> Hidroxilamina hidrocloruro </td><td> ThermoFisher </td><td> H330 </td><td></td></tr><tr><td> 5&#39;-difosfato de adenosina sal de sodio </td><td> Sigma Aldrich </td><td> A2754 </td><td></td></tr><tr><td> BIOMATE III Espectrofotómetro </td><td> ThermoFisher </td><td> 142982082 </td><td> Estándar de UV / Vis espectrofotómetro </td></tr></table>

direct detection of the acetate-forming activity  of the enzyme acetate kinase

Quinasa de acetato, un miembro del acetato y el azúcar-quinasa Hsp70-actina (ASKHA) superfamilia de enzimas 1-5, es el responsable de la fosforilación reversible de acetato de acetil fosfato que utilizan ATP como sustrato. Acetato de quinasas son muy abundantes en las bacterias, que se encuentra en un género de Archaea, y también están presentes en los microbios de la Eukarya 6. El acetato quinasa mejor caracterizados es que a partir del metano que producen thermophila archaeon Methanosarcina 14.07. Una quinasa acetato de que sólo se puede utilizar PP i ATP, pero no en la dirección de la acetil-fosfato forman se ha aislado de Entamoeba histolytica, el agente causante de la disentería amebiana, y hasta el momento sólo se han encontrado en este género 15,16. En la dirección de la formación de acetil fosfato, acetato de actividad quinasa se mide típicamente usando el ensayo de hidroxamato, describió por primera vez por Lipmann17-20, un ensayo acoplado en lo que se suma la conversión de ATP a ADP a la oxidación de NADH a NAD + por la piruvato quinasa enzimas lactato deshidrogenasa y 21,22, o un ensayo de medición de la liberación de fosfato inorgánico después de la reacción del producto acetil fosfato con hidroxilamina 23. Actividad en la dirección opuesta, el acetato de formación de la dirección se mide mediante el acoplamiento de la formación de ATP a partir de ADP a la reducción de NADP + a NADPH por las enzimas hexoquinasa y glucosa 6-fosfato deshidrogenasa 24. Aquí se describe un método para la detección de la actividad quinasa de acetato en la dirección de la formación de acetato que no requiere de enzimas de acoplamiento, sino que se basa en la determinación directa del consumo de acetil fosfato. Después de la reacción enzimática, fosfato restante acetil se convierte en un complejo hidroxamato férrico que se puede medir espectrofotométricamente, como para el ensayo de hidroxamato. Por lo tanto, a diferencia del stensayo andard junto a esta dirección, que depende de la producción de ATP a partir de ADP, este ensayo directo se pueden utilizar para las quinasas de acetato que producen ATP o PP i.

Watch this Scientific Journal Video about Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase at JoVE.com

Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

Un método para la determinación de la actividad quinasa de acetato se describe. Este ensayo utiliza una reacción directa para determinar la actividad enzimática y la cinética de la cinasa de acetato en la dirección de acetato de formación con diferentes aceptores fosforilo. Además, este método puede ser utilizado para el ensayo de fosfato de otros o acetil de acetil-CoA utilizando enzimas.

La detección directa de la actividad de acetato de formación de la enzima cinasa de acetato

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of ...

direct-detection-acetate-forming-activity-enzyme-acetate

Research

JoVE Journal

Biology

23.0K vistas.  Clemson University. Un método para la determinación de la actividad quinasa de acetato se describe. Este ensayo utiliza una reacción directa para determinar la actividad enzimática y la cinética de la cinasa de acetato en la dirección de acetato de formación con diferentes aceptores fosforilo. Además, este método puede ser utilizado para el ensayo de fosfato de otros o acetil de acetil-CoA utilizando enzimas.

Video: Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

La detección de ubiquitinación de proteínas

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Investigación

Biología

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 ...

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform.
Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5.
The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution.
At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution.
A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

A protocol to detect trichothecenes (mycotoxins of concern for human health) using a newly developed screening method based on a competitive immunochemical method and a final electrochemical detection is demonstrated.

ELIME (Enzyme Linked Inmuno magnética químicos) Método para la detección de micotoxinas

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

La producción de transgénicos Xenopus laevis Integración mediada por la enzima de restricción y transferencia nuclear

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

La detección de óxido nítrico y el radical anión superóxido por espectroscopía de resonancia paramagnética de electrones de las células utilizando atrapadores

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

Seguimiento de las actividades de quinasa y fosfatasa través del ciclo celular por Proporcional FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

Ensayo de la actividad quinasa de LRRK2 In vitro</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. Current strategies for microbial analysis include both traditional laboratory culture-dependent techniques and those based on direct extraction and determination of certain biomarkers1, 2. Few among the diversity of microbial species inhabiting soil can be cultured, so culture-dependent methods introduce significant biases, a limitation absent in biomarker analysis.
The glucosamine, mannosamine, galactosamine and muramic acid have been well served as measures of both the living and dead microbial mass, of these the glucosamine (most abundant) and muramic acid (uniquely from bacterial cell) are most important constituents in the soil systems3, 4. However, the lack of knowledge on the analysis restricts the wide popularization among scientific peers. Among all existing analytical methods, derivatization to aldononitrile acetates followed by GC-based analysis has emerged as a good option with respect to optimally balancing precision, sensitivity, simplicity, good chromatographic separation, and stability upon sample storage5.
Here, we present a detailed protocol for a reliable and relatively simple analysis of glucosamine and muramic acid from soil after their conversion to aldononitrile acetates. The protocol mainly comprises four steps: acid digestion, sample purification, derivatization and GC determination. The step-by-step procedure is modified according to former publications6, 7. In addition, we present a strategy to structurally validate the molecular ion of the derivative and its ion fragments formed upon electron ionization. We applied GC-EI-MS-SIM, LC-ESI-TOF-MS and isotopically labeled reagents to determine the molecular weight of aldononitrile acetate derivatized glucosamine and muramic acid; we used the mass shift of isotope-labeled derivatives in the ion spectrum to investigate ion fragments of each derivatives8. In addition to the theoretical elucidation, the validation of molecular ion of the derivative and its ion fragments will be useful to researchers using &delta;13C or ion fragments of these biomarkers in biogeochemical studies9, 10.

We describe a method protocol for the GC-based analysis of the aldonitrile acetate derivatives of glucosamine and muramic acid extracted from soil. For elucidation of the chemical mechanism, we also present a strategy to confirm the structure of the derivative and the ion fragments formed upon electron ionization.

GC-basado en la detección de ácido Aldononitrile acetato derivado de glucosamina y murámico de la determinación de residuos microbiana en el suelo

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

La medición de las larvas La actividad en el<em&gt; Drosophila</em&gt; Monitor de Actividad

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

Studying the Activity of Neuropeptides and Other Regulators of the Excretory System in the Adult Mosquito

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the foundation to develop novel strategies for pest control. Here, a series of methods are described that are routinely utilized to determine the functional roles of neuropeptides and other neuronal factors (i.e., biogenic amines) on the excretory system of the mosquito, Aedes aegypti. The Malpighian tubules (MTs), responsible for primary urine formation, can continue functioning for hours when removed from the mosquito, allowing for fluid secretion measurements following hormone treatments. As such, the Ramsay assay is a useful technique to measure secretion rates from isolated MTs. Ion-selective microelectrodes (ISME) can sequentially be used to measure ion concentrations (i.e., Na+ and K+) in the secreted fluid. This assay allows for the measurement of several MTs at a given time, determining the effects of various hormones and drugs. The Scanning Ion-selective Electrode Technique uses ISME to measure voltage representative of ionic activity in the unstirred layer adjacent to the surface of ion transporting organs to determine transepithelial transport of ions in near real time. This method can be used to understand the role of hormones and other regulators on ion absorption or secretion across epithelia. Hindgut contraction assays are also a useful tool to characterize myoactive neuropeptides, that may enhance or reduce the ability of this organ to remove excess fluid and waste. Collectively, these methods provide insight into how the excretory system is regulated in adult mosquitoes. This is important because functional coordination of the excretory organs is crucial in overcoming challenges such as desiccation stress after eclosion and before finding a suitable vertebrate host to obtain a bloodmeal.

This protocol outlines methodologies behind the Ramsay assay, ion-selective microelectrodes, Scanning Ion-selective Electrode Technique (SIET) and in vitro contraction assays, applied to study the adult mosquito excretory system, comprised of the Malpighian tubules and hindgut, to collectively measure ion and fluid secretion rates, contractile activity, and transepithelial ion transport.

Estudio de la actividad de los neuropéptidos y otros reguladores del sistema excretor en el mosquito adulto

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the ...