De voorbereiding van Synaptoneurosomes van Mouse Cortex met behulp van een Discontinue Percoll-Sucrose dichtheidsgradiënt
Summary
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.
Abstract
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.
Protocol
Discussion
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 …
Disclosures
The authors have nothing to disclose.
Acknowledgements
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).
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
|---|---|---|---|
| Micro BCA Protein Assay Kit | Pierce | 23235 | |
| CaCl2 | Fisher | C79-500 | |
| CO2 gas | Airgas (UW-MDS) | CD 50 | |
| EDTA | RPI | E57020 | |
| EtOH | Fisher | A407SK-4 | |
| HCl | Fisher | A142-212 | |
| Percoll | GE Healthcare | 17-0891-01 | |
| KH2PO4 | Fisher | P285-500 | |
| PierceSDS-PAGE Sample Prep Kit | Pierce | 89888 | |
| NaCl | RPI | S23020 | |
| NaHCO3 | Fisher | BP328-500 | |
| Na2PHO4 | Fisher | S381-500 | |
| Sucrose | RPI | S24060 | |
| Tris Base | RPI | T60040 | |
| Tetrodotoxin | Sigma | T5651 | |
| Express Pro Label Mix S35 Easy Tag | Perkin Elmer | NEG772 | |
| Equipment | Company | Catalogue number | Comments (optional) |
| Dissection tools | |||
| Dounce homogenizer, 7 mL (comes with two glass pestles labled ” A” and “B”) | Wheaton | ||
| P1000 Gilson Pipetman | Gilson | F123602 | |
| Allegra 6KR Centrifuge | Beckman Coulter | 366830 | |
| GH 3.8 Rotor, Swinging bucket rotor | Beckman Coulter | 360581 | |
| Beckman J2-21 Centrifuge | Beckman | ||
| Beckman tubes with caps | Beckman | 355672 | |
| White walled adapters | Beckman | 342327 | |
| Blue walled adapters | Beckman | ||
| JA-17 Rotor, Fixed-angle rotor | Beckman | 369691 |
Table of antibodies used for western blots:
| Name of Antibody | Company | Catalogue number | Host Species | Dilution Factor |
|---|---|---|---|---|
| β-Actin | Sigma | A5441 | mouse | 1:2000 |
| GFAP | Santa Cruz | sc-65343 | mouse | 1:200 |
| GP73 | Santa Cruz | Sc-134509 | rabbit | 1:200 |
| HSC70 | Santa Cruz | sc-7298 | mouse | 1:200 |
| Laminβ | Santa Cruz | Sc-6261 | goat | 1:200 |
| Prohibitin | Santa Cruz | sc-28259 | rabbit | 1:200 |
| PSD95 | Millipore | MAB1596 | mouse | 5 μg/μL |
| SNAP25 | AbCam | ab5666-100 | rabbit | 1:2000 |
| Synaptophysin | Millipore | MAB368 | mouse | 1:500 |
| β-3-Tubulin | Santa Cruz | sc-80016 | mouse | 1:200 |
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