Name: preparation of synaptoneurosomes from mouse cortex using a discontinuous percoll-sucrose density gradient
Uploaded: 2011-09-17T14:00:00
Description: Watch this Scientific Journal Video about Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient at JoVE.com

Gostaríamos de agradecer a BK agosto da Universidade de Wisconsin-Madison Facility Microscópio Eletrônico para a microscopia eletrônica. Este trabalho foi financiado pelo NIH concede R01-DA026067 e P30-HD03352 (a JSM).

1. Preparações 1-4 <ol><li> Prepare 500 mL de tampão de meio de gradiente (GM), misturando 50 mL de uma pasta de sacarose 2,5 M, 2,5 mL de uma 1M Tris-HCl, pH 7,5 valores, e 0,1 mL de um 0,5 m EDTA, pH 8,0 estoque com mili- Q água para volume. Filtro de esterilizar a solução de alíquota, e armazenar congelados. </li><li> Prepare um estoque de 1000x tetrodotoxina (TTX) fazendo uma solução de 1 mM em mili-Q H 2 O. Alíquotas de TTX podem ser armazenadas a -20 ° C. </li><li> Preparar o tam...

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A preparação de gradiente descontínuo de Percoll-sacarose aqui descrito é um método rápido e confiável para isolar SNs ativo, que pode ser usado em uma variedade de experimentos de transmissão sináptica. Este método do gradiente, que é baseado no método desenvolvido por Dunkley et al., 3,4 é um procedimento de fracionamento subcelular cérebro que isola pré e pós-sináptica da membrana derivada vesículas que estão associados com o outro. Sem purificação adicional esses SNs são compatíveis...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> Micro BCA Protein Assay Kit </td><td> Perfurar </td><td> 23235 </td><td></td></tr><tr><td> CaCl 2 </td><td> Pescador </td><td> C79-500 </td><td></td></tr><tr><td> Gás CO2 </td><td> Airgas (UW-MDS) </td><td> CD 50 </td><td></td></tr><tr><td> EDTA </td><td> RPI </td><td> E57020 </td><td></td></tr><tr><td> EtOH </td><td> Pescador </td><td> A407SK-4 </td><td></td></tr><tr><td> HCl </td><td> Pescador </td><td> A142-212 </td><td></td></tr><tr><td> Percoll </td><td> GE Healthcare </td><td> 17-0891-01 </td><td></td></tr><tr><td> KH 2 PO 4 </td><td> Pescador </td><td> P285-500 </td><td></td></tr><tr><td> PierceSDS PAGE-Sample Prep Kit </td><td> Perfurar </td><td> 89888 </td><td></td></tr><tr><td> NaCl </td><td> RPI </td><td> S23020 </td><td></td></tr><tr><td> NaHCO 3 </td><td> Pescador </td><td> BP328-500 </td><td></td></tr><tr><td> Na 2 PHO 4 </td><td> Pescador </td><td> S381-500 </td><td></td></tr><tr><td> Sacarose </td><td> RPI </td><td> S24060 </td><td></td></tr><tr><td> Tris Base de Dados </td><td> RPI </td><td> T60040 </td><td></td></tr><tr><td> Tetrodotoxina </td><td> Sigma </td><td> T5651 </td><td></td></tr><tr><td> Expressar Etiqueta Pro Mix S35 Tag Fácil </td><td> Perkin Elmer </td><td> NEG772 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><th> Equipamento </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> Ferramentas de dissecção </td><td></td><td></td><td></td></tr><tr><td> Dounce homogeneizador, 7 mL (vem com dois pilões de vidro &quot;A&quot; e &quot;B&quot;) </td><td> Wheaton </td><td></td><td></td></tr><tr><td> P1000 Gilson Pipetman </td><td> Gilson </td><td> F123602 </td><td></td></tr><tr><td> Allegra Centrifugar 6KR </td><td> Beckman Coulter </td><td> 366830 </td><td></td></tr><tr><td> GH Rotor 3.8, rotor de caçamba móvel </td><td> Beckman Coulter </td><td> 360581 </td><td></td></tr><tr><td> Centrífuga Beckman J2-21 </td><td> Beckman </td><td></td><td></td></tr><tr><td> Beckman tubos com tampas </td><td> Beckman </td><td> 355672 </td><td></td></tr><tr><td> Branco adaptadores murada </td><td> Beckman </td><td> 342327 </td><td></td></tr><tr><td> Azul adaptadores murada </td><td> Beckman </td><td></td><td></td></tr><tr><td> JA-17 Rotor, rotor de ângulo fixo </td><td> Beckman </td><td> 369691 </td><td></td></tr></tbody></table> Tabela de anticorpos utilizados para western blots: <table border="1"><tbody><tr><th> Nome de anticorpos </th><th> Companhia </th><th> Número de catálogo </th><th> Espécies hospedeiras </th><th> Fator de diluição </th></tr><tr><td> β-Actina </td><td> Sigma </td><td> A5441 </td><td> mouse </td><td> 1:2000 </td></tr><tr><td> GFAP </td><td> Santa Cruz </td><td> sc-65343 </td><td> mouse </td><td> 1:200 </td></tr><tr><td> GP73 </td><td> Santa Cruz </td><td> Sc-134509 </td><td> coelho </td><td> 1:200 </td></tr><tr><td> HSC70 </td><td> Santa Cruz </td><td> sc-7298 </td><td> mouse </td><td> 1:200 </td></tr><tr><td> Laminβ </td><td> Santa Cruz </td><td> Sc-6261 </td><td> cabra </td><td> 1:200 </td></tr><tr><td> Prohibitin </td><td> Santa Cruz </td><td> sc-28259 </td><td> coelho </td><td> 1:200 </td></tr><tr><td> PSD95 </td><td> Millipore </td><td> MAB1596 </td><td> mouse </td><td> 5 mg / mL </td></tr><tr><td> SNAP25 </td><td> Abcam </td><td> ab5666-100 </td><td> coelho </td><td> 1:2000 </td></tr><tr><td> Sinaptofisina </td><td> Millipore </td><td> MAB368 </td><td> mouse </td><td> 1:500 </td></tr><tr><td> β-3-tubulina </td><td> Santa Cruz </td><td> sc-80016 </td><td> mouse </td><td> 1:200 </td></tr></tbody></table>

preparation of synaptoneurosomes from mouse cortex using a discontinuous percoll-sucrose density gradient

Synaptoneurosomes (SNS) são obtidos após homogeneização e fracionamento de rato córtex cerebral. Eles são selados vesículas ou terminais isolados que romper com os terminais do axônio quando o tecido cortical é homogeneizado. Os SNs reter características pré e pós-sináptico, o que os torna útil no estudo da transmissão sináptica. Conservem as máquinas moleculares utilizados na sinalização neuronal e são capazes de captação, armazenamento e liberação de neurotransmissores. A produção e isolamento de ativos SNs pode ser problemático mídias usando como Ficoll, que podem ser citotóxicos e centrifugação prolongada devido a alta densidade, e filtração e métodos de centrifugação, que pode resultar em baixa atividade, devido a danos mecânicos do SNS. No entanto, o uso de gradientes descontínuos de Percoll-sacarose de densidade para isolar SNs fornece um método rápido para produzir bons rendimentos de SNs traducionalmente ativo. O método do gradiente Percoll-sacarose é rápido e gentil como ele emprega condições isotônica, tem spins centrifugação menos e mais curtos e evita que as etapas de centrifugação pellet SNs e causar danos mecânicos.

Watch this Scientific Journal Video about Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient at JoVE.com

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Um método para preparar traducionalmente ativa, synaptoneurosomes intacta (SNs) de córtex cerebral de rato é descrito. O método utiliza um gradiente de densidade descontínua Percoll-sacarose permitindo a preparação rápida de ativos SNs.

Preparação de Synaptoneurosomes de rato Cortex usando um gradiente de densidade Percoll descontínuo Sacarose-

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

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In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Imagem Funcional do Cortex Auditivo em gatos adultos usando de alta campo fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Imagens ao vivo da mitose no desenvolvimento Cortex embrionárias de camundongos

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Two-Photon<em&gt; In vivo</em&gt; Imagem de espinhas dendríticas no Rato Cortex Usando um desbaste-crânio Preparação

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

Preparação do Synaptic Plasma Membrane e Proteínas de densidade pós-sinápticos Usando um gradiente de sacarose descontínuo

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in ...

Investigating the Function of Deep Cortical and Subcortical Structures Using Stereotactic Electroencephalography: Lessons from the Anterior Cingulate Cortex

Investigar a função do Deep Cortical e estruturas subcorticais Usando estereotáxica Eletroencefalografia: Lições da Cortex cingulado anterior

Stereotactic Electroencephalography (SEEG) is a technique used to localize seizure foci in patients with medically intractable epilepsy. This procedure ...