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Neuroscience

Süreksiz Percoll Sakkaroz Yoğunluk Gradient kullanarak Fare Cortex Synaptoneurosomes hazırlanması

Published: September 17, 2011
doi:
10.3791/3196
, , ,
1Department of Pathology and Laboratory Medicine, Waisman Center for Developmental Disabilities,University of Wisconsin, 2Department of Biochemistry, Waisman Center for Developmental Disabilities,University of Wisconsin

Summary

Fare beyin korteksi translationally etkin, sağlam synaptoneurosomes (SNS) hazırlamak için bir yöntem açıklanmıştır. Bu yöntem, aktif SNS hızlı hazırlanması için izin veren bir süreksiz Percoll sakaroz yoğunluk gradiyenti kullanır.

Abstract

Synaptoneurosomes (SNS) fare beyin korteksi homojenizasyon ve bölümleme sonra elde edilir. Bunlar kortikal doku homojenize akson terminalleri kurtulmak vezikül veya izole terminalleri kapatılmalı. SNS sinaptik iletim çalışmasında faydalı kılan, pre-ve postsinaptik özelliklerini korurlar. Nöronal sinyal kullanılan moleküler makine korumak ve alımı, depolama ve nörotransmitterlerin salınımı yeteneğine sahiptir.

Aktif SNS üretim ve izolasyon sitotoksik ve SNS mekanik hasar nedeniyle düşük aktivite neden olabilir, yüksek yoğunluklu ve filtrasyon ve santrifüj yöntemleri nedeniyle genişletilmiş santrifüj gerektirebilir Ficoll gibi sorunlu kullanarak medias olabilir. Ancak, süreksiz Percoll sakaroz yoğunluğu gradyanlar SNS izole etmek için kullanmak translationally aktif SNS iyi verim üretmek için hızlı bir yöntem sağlar. Percoll sukroz gradient yöntemi, hızlı ve nazik bir pelet SNS ve mekanik hasara neden olduğu, izotonik koşullar istihdam ve daha az ve daha kısa santrifüj spin santrifüj adımları önler.

Protocol

1. Hazırlıklar 1-4 50 ml 2.5 M sakkaroz bulamaç karıştırılarak 500 mL (GM), degrade orta tampon hazırlayın, 1M Tris-HCl, pH7.5 hisse senedi ve 0,5 m EDTA 0.1 ml 2.5 ml, pH 8.0, stok Mili S su hacmi. Çözümü, kısım ve mağaza dondurulmuş sterilize edin Filtre. Mili-Q H 2 O. 1 mM çözüm yaparak tetrodotoxin (TTX) 1000x stok hazırlayın TTX alikotları -20 ° C'de saklanabilir Mili Q suyu 100 mM Tris (pH 7.5), 5 mM Na 2 HPO 4, …

Discussion

Burada açıklanan süreksiz Percoll sukroz gradient hazırlık sinaptik iletim deneyler çeşitli kullanılabilir aktif SNS, izole etmek için hızlı ve güvenilir bir yöntemdir. Dunkley ve ark. 3,4 tarafından geliştirilen yöntem dayanmaktadır Bu gradyan yöntemi, birbiri ile ilişkili hem de pre-ve postsinaptik membran kaynaklı veziküller izole bir hücre içi beyin fraksiyonasyon işlemdir. Ileri arıtma olmadan bu SNS immunoprecipitation, batı blot, flow sitometri, [35S] üzerinden Met dahil, ele…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Biz, elektron mikroskobu için, Wisconsin-Madison Elektron Mikroskobu Tesisi Üniversitesi'nden BK Ağustos teşekkür etmek istiyorum. Bu çalışma NIH hibe R01-DA026067 ve P30-HD03352 (JSM) tarafından desteklenmiştir.

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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Tags

SynaptoneurosomesMouse CortexDiscontinuous Percoll-Sucrose Density GradientHomogenizationFractionationResealed VesiclesIsolated TerminalsSynaptic TransmissionPre- And Postsynaptic CharacteristicsNeuronal SignalingNeurotransmittersFicollCytotoxicCentrifugationFiltrationMechanical DamagePercoll-sucrose Gradient MethodTranslationally Active SNsIsotonic Conditions
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Westmark, P. R., Westmark, C. J., Jeevananthan, A., Malter, J. S. Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient. J. Vis. Exp. (55), e3196, doi:10.3791/3196 (2011).
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