Name: protein misfolding cyclic amplification of prions
Uploaded: 2012-11-07T12:00:00
Description: Watch this Scientific Journal Video about Protein Misfolding Cyclic Amplification of Prions at JoVE.com

Nos gustaría dar las gracias a la Dra. Marie Ramos Vesper Fe para la lectura crítica del manuscrito. Este trabajo fue apoyado por el Centro Nacional para Recursos de Investigación (P20 RR0115635-6, C06 RR17417-01 y G20RR024001) y el Instituto Nacional de Trastornos Neurológicos y Accidentes Cerebrovasculares (2R01 NS052609).

1. Preparación del Equipo <ol><li> Utilice un Misonix 3000 o 4000 sonicador Misonix (Farmingdale, NY) conectado a un baño de Thermo Electron Neslab EX-7 agua (Newington, NH) para mantener una temperatura constante de 37 ° C. Sonicar las muestras en 200 ul de paredes delgadas tiras de tubos de PCR con tapas abovedadas obtenidos de Thermo Scientific (Waltham, MA). </li><li> Un baño de ultrasonidos nueva marca requiere de un &quot;robo&quot; período de operación continua 9. Un robo de dos meses consiste ...

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enzymatic+treatment+of+soil-bound+prions+effectively+inhibits+replication.">An enzymatic treatment of soil-bound prions effectively inhibits replication.</a> Appl. Environ. Microbiol. 77, 4313-4317 (2011).</li><li>Saunders, S. E., Shikiya, R. A., Langenfeld, K., Bartelt-Hunt, S. L., Bartz, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+efficiency+of+soil-bound+prions+varies+with+soil+type.">Replication efficiency of soil-bound prions varies with soil type.</a> Journal of virology. , (2011).</li><li>Schutt, C. R., Bartz, J. 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Desafíos de la amplificación de las proteínas priónicas infecciosas son los períodos de incubación largos y los gastos de los experimentos in vivo. La técnica EPCP es un medio rentable para amplificar los agentes infecciosos de priones. Varios laboratorios han confirmado la capacidad de PMCA para amplificar con precisión las cepas de priones in vitro 7, 9, 12, 14, 19,24. Las enfermedades por priones se pueden transmitir entre especies. Bessen y M...

<table border="1"><tbody><tr><td> Reactivo / Equipo </td><td> Fabricante </td><td> Cat. Número </td></tr><tr><td> Misonix 3000 </td><td> Misonix </td><td> S-3000 </td></tr><tr><td> Misonix 4000 </td><td> Misonix </td><td> S-4000 </td></tr><tr><td> Tenbroeck triturador de tejidos </td><td> Kontes </td><td> 885000-0007 </td></tr><tr><td> Neslab EX-7 Water Bath </td><td> Thermo Electron </td><td> Neslab EX-7 </td></tr><tr><td> PCR de 0,2 ml tiras de tubos </td><td> Thermo Scientific </td><td> AB-0451 </td></tr><tr><td> Triton X-100 </td><td> Sigma Aldrich </td><td> T9284-100ML </td></tr><tr><td> Inhibidor de Proteasa Completo </td><td> Roche </td><td> 11 697 498 001 </td></tr><tr><td> EDTA </td><td> JT Baker </td><td> 4040-00 </td></tr><tr><td> DPBS </td><td> Mallinckrodt Baker Mediatech </td><td> 21-031-CV </td></tr><tr><td> Versi-Dry Lab Soakers </td><td> Fisher Scientific </td><td> 14 206 28 </td></tr><tr><td> Repti Therm calentador </td><td> Zoo Med Laboratories, Inc. </td><td> RH-4 </td></tr></tbody></table>

protein misfolding cyclic amplification of prions

Los priones son los agentes infecciosos causantes de la encefalopatía espongiforme transmisible inevitablemente fatal (EET) en los animales y los seres humanos 9,18. La proteína prión tiene dos isoformas distintas, la no infecciosa proteína codificada por el hospedador (PrP C) y la proteína infecciosa (PrP Sc), una isoforma anormal de PrP plegado en C 8. Uno de los retos de trabajar con agentes de priones es el largo período de incubación antes de la aparición de los signos clínicos después de la inoculación de acogida 13. Este mandato tradicionalmente los estudios en animales largos y caros de bioensayo. Además, las propiedades bioquímicas y biofísicas de PrP Sc están pobremente caracterizados debido a su conformación inusual y estados de agregación. PrP Sc puede sembrar la conversión de PrP C y PrP Sc in vitro 14. El EPCP es una técnica in vitro que se Advantage de esta capacidad utilizando ciclos de sonicación y la incubación para producir grandes cantidades de PrP Sc, a un ritmo acelerado, a partir de un sistema que contiene cantidades en exceso de PrP C y pequeñas cantidades de la semilla PrP Sc 19. Esta técnica ha demostrado ser eficaz para recapitular la especificidad de especie y cepa de PrP Sc conversión de PrP C, para emular interferencia cepa de priones, y para amplificar los niveles muy bajos de PrP Sc partir de tejidos infectados, fluidos y muestras ambientales 6,7,16, 23. Este documento detalla el protocolo EPCP, incluyendo recomendaciones para minimizar la contaminación, generando resultados consistentes, y la cuantificación de los resultados. También se discuten varias aplicaciones del EPCP, incluyendo la generación y caracterización de cepas de priones infecciosos, la interferencia cepa de prión, y la detección de priones en el medio ambiente.

Misfolding proteína amplificación cíclica (PMCA) se utiliza para amplificar PrP Sc in vitro 7, 12, 14, 19, 24. El éxito de la amplificación PrP Sc se muestra por un aumento en la intensidad de la banda en las transferencias Western de la proteína priónica PK-resistente (migrando entre 19 y 30 kDa para las cepas de priones de hámster derivados) como se muestra en la Figura 3. El aumento de la intensidad de la banda después de su EPCP indica la amplificac...

Watch this Scientific Journal Video about Protein Misfolding Cyclic Amplification of Prions at JoVE.com

Protein Misfolding Cyclic Amplification of Prions

Misfolding proteína amplificación cíclica (EPCP) es un ensayo in vitro para el estudio de la conversión de priones y barreras cepa y especie. También se puede utilizar como un ensayo de detección de priones.

Mal plegamiento de proteínas priones de amplificación cíclica

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

Research

JoVE Journal

Immunology and Infection

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Un enfoque comparativo para caracterizar el paisaje del Huésped-Patógeno interacciones proteína-proteína

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Investigación

Inmunología e Infección

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Procedimientos para la Identificación de priones infecciosos después de pasar por el sistema digestivo de una especie de aves

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Demostración de un resistente a múltiples fármacos<em&gt; Mycobacterium tuberculosis</em&gt; Microarray Amplificación

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Isotérmico de amplificación (LAMP) Ensayos Loop mediada para la detección específica de las especies de<em&gt; Eimeria</em&gt; Que infectan pollos

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

El desarrollo de la mediación de la Loop liofilizada amplificación isotérmica reactivos para la detección de<em&gt; Coxiella burnetii</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

En tiempo real temblando inducida por conversión de ensayo para la detección de priones CWD en materia Fecal

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant ...

Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers

Construcción de una plataforma de proyección de alto rendimiento para evaluar la heterogeneidad de la amplificación del gen HER2 en cáncer de mama

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

Disección señalización complejos por purificación de la afinidad de complementación Bimolecular (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Análisis conductual de gran C. elegans las poblaciones utilizando un amplio campo de visión de seguimiento plataforma automatizada

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

Generación, amplificación y titulación de virus sincitiales respiratorios recombinantes

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...