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

We willen graag BK augustus bedanken van de Universiteit van Wisconsin-Madison Electron Microscope faciliteit voor de elektronenmicroscopie. Dit werk werd ondersteund door NIH subsidie ​​R01-DA026067 en P30-HD03352 (tot JSM).

1. Voorbereidingen 1-4 <ol><li> Bereid 500 ml gradiënt medium (GM) buffer door het mengen van 50 ml van een 2,5 M sucrose slurry, 2,5 ml van een 1 M Tris-HCl, pH 7,5 voorraad, en 0,1 ml van een 0,5 m EDTA, pH 8,0 voorraad met milli- Q water om het volume. Filter steriliseren van de oplossing, hoeveelheid en opslaan van bevroren. </li><li> Bereid een 1000x voorraad tetrodotoxine (TTX) door het maken van een een mM oplossing in milli-Q H 2 O. Aliquots van TTX kan worden opgeslagen bij -20 ° C. </...

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De discontinue Percoll-sucrose gradiënt bereiding hierin beschreven is een snelle, betrouwbare methode om actief SNS, die gebruikt kunnen worden in een verscheidenheid van synaptische transmissie experimenten te isoleren. Dit verloop methode, die is gebaseerd op de methode ontwikkeld door Dunkley et al.., 3,4 is een subcellulaire hersenen fractionering procedure, die zowel pre-en postsynaptische membraan-afgeleide blaasjes die geassocieerd zijn met elkaar isoleert. Zonder verdere zuivering van deze SNS zijn ...

<table border="1"><tbody><tr><th> Naam van het serum </th><th> Vennootschap </th><th> Catalogus nummer </th><th> Commentaar (optioneel) </th></tr><tr><td> Micro BCA Protein Assay Kit </td><td> Doorboren </td><td> 23235 </td><td></td></tr><tr><td> CaCl 2 </td><td> Visser </td><td> C79-500 </td><td></td></tr><tr><td> CO 2-gas </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> Visser </td><td> A407SK-4 </td><td></td></tr><tr><td> HCl </td><td> Visser </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> Visser </td><td> P285-500 </td><td></td></tr><tr><td> PierceSDS-PAGE Sample Prep Kit </td><td> Doorboren </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> Visser </td><td> BP328-500 </td><td></td></tr><tr><td> Na 2 PHO 4 </td><td> Visser </td><td> S381-500 </td><td></td></tr><tr><td> Sucrose </td><td> RPI </td><td> S24060 </td><td></td></tr><tr><td> Tris Base </td><td> RPI </td><td> T60040 </td><td></td></tr><tr><td> Tetrodotoxine </td><td> Sigma </td><td> T5651 </td><td></td></tr><tr><td> Express Pro Label Mix S35 Gemakkelijk Tag </td><td> Perkin Elmer </td><td> NEG772 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><th> Uitrusting </th><th> Vennootschap </th><th> Catalogus nummer </th><th> Commentaar (optioneel) </th></tr><tr><td> Dissectie-instrumenten </td><td></td><td></td><td></td></tr><tr><td> Dounce homogenisator, 7 ml (wordt geleverd met twee glazen stampers met het label &quot;A&quot; en &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 6KR Centrifuge </td><td> Beckman Coulter </td><td> 366830 </td><td></td></tr><tr><td> GH 3,8 Rotor, Swinging emmer rotor </td><td> Beckman Coulter </td><td> 360581 </td><td></td></tr><tr><td> Beckman J2-21 Centrifuge </td><td> Beckman </td><td></td><td></td></tr><tr><td> Beckman buizen met doppen </td><td> Beckman </td><td> 355672 </td><td></td></tr><tr><td> Wit ommuurde adapters </td><td> Beckman </td><td> 342327 </td><td></td></tr><tr><td> Blauw ommuurde adapters </td><td> Beckman </td><td></td><td></td></tr><tr><td> JA-17 Rotor, vaste-hoek rotor </td><td> Beckman </td><td> 369691 </td><td></td></tr></tbody></table> Tabel van antilichamen gebruikt worden voor western blots: <table border="1"><tbody><tr><th> Naam van het antilichaam </th><th> Vennootschap </th><th> Catalogus nummer </th><th> Gastheersoort </th><th> Verdunningsfactor </th></tr><tr><td> β-actine </td><td> Sigma </td><td> A5441 </td><td> muis </td><td> 1:2000 </td></tr><tr><td> GFAP </td><td> Santa Cruz </td><td> sc-65343 </td><td> muis </td><td> 1:200 </td></tr><tr><td> GP73 </td><td> Santa Cruz </td><td> Sc-134509 </td><td> konijn </td><td> 1:200 </td></tr><tr><td> HSC70 </td><td> Santa Cruz </td><td> sc-7298 </td><td> muis </td><td> 1:200 </td></tr><tr><td> Laminβ </td><td> Santa Cruz </td><td> Sc-6261 </td><td> geit </td><td> 1:200 </td></tr><tr><td> Prohibitin </td><td> Santa Cruz </td><td> sc-28259 </td><td> konijn </td><td> 1:200 </td></tr><tr><td> PSD95 </td><td> Millipore </td><td> MAB1596 </td><td> muis </td><td> 5 ug / ul </td></tr><tr><td> SNAP25 </td><td> AbCam </td><td> ab5666-100 </td><td> konijn </td><td> 1:2000 </td></tr><tr><td> Synaptofysine </td><td> Millipore </td><td> MAB368 </td><td> muis </td><td> 1:500 </td></tr><tr><td> β-3-tubuline </td><td> Santa Cruz </td><td> sc-80016 </td><td> muis </td><td> 1:200 </td></tr></tbody></table>

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

Synaptoneurosomes (SNS) zijn verkregen na homogenisering en fractionering van muizenhersenen cortex. Ze worden opnieuw verzegeld blaasjes of geïsoleerd terminals die los te maken van axon terminals als de corticale weefsel wordt gehomogeniseerd. De SNS behoudt pre-en postsynaptische eigenschappen, waardoor ze nuttig zijn in de studie van de synaptische transmissie. Zij behouden de moleculaire machines gebruikt in de neuronale signalering en zijn in staat van opname, opslag en afgifte van neurotransmitters. De productie en isolatie van actieve SNS kan problemen opleveren met behulp van media, zoals Ficoll, die kunnen worden cytotoxische en vereisen uitgebreid centrifugeren door de hoge dichtheid, en filtratie en centrifugeren methoden, die kan in lage activiteit resultaat als gevolg van mechanische schade van de SNS. Echter, het gebruik van discontinue Percoll-sucrose dichtheidsgradiënten aan SNS isoleren biedt een snelle methode om een ​​goede opbrengst van translationeel actief SNS produceren. De Percoll-sucrose gradiënt methode is snel en zacht als zij in dienst isotone omstandigheden, heeft minder en kortere centrifugeren spins en vermijdt centrifugeren stappen die pellet SNS en mechanische schade.

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

Een methode om translationeel actief, intact synaptoneurosomes (SNS) voor te bereiden van de hersenen van muizen cortex wordt beschreven. De methode maakt gebruik van een discontinue Percoll-sucrose dichtheidsgradiënt waardoor voor de snelle bereiding van actieve SNS.

De voorbereiding van Synaptoneurosomes van Mouse Cortex met behulp van een Discontinue Percoll-Sucrose dichtheidsgradiënt

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

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

Functionele Beeldvorming van de auditieve cortex in volwassen katten met behulp van High-field 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

Live-Imaging van mitose in het ontwikkelen van muis embryonale Cortex

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

Twee-Photon<em&gt; In vivo</em&gt; Imaging van vertakte stekels in de Mouse Cortex met een uitgedunde schedel Voorbereiding

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

Voorbereiding van Synaptic plasmamembraan en Postsynaptische Density eiwitten met behulp van een discontinue sucrosegradiënt

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

Bestudering van de functie van Deep Corticale en subcorticale structuren met behulp van stereotactische Electroencephalography: Lessen uit de anterior cingulate cortex

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