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Neuroscience

माउस प्रांतस्था से Synaptoneurosomes असंतत घनत्व Percoll Sucrose ढाल का उपयोग कर की तैयारी

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

माउस मस्तिष्क प्रांतस्था से translationally सक्रिय बरकरार synaptoneurosomes (SNS) तैयार करने की विधि वर्णित है. विधि एक असंतत घनत्व Percoll – sucrose सक्रिय SNS की जल्दी तैयार करने के लिए अनुमति देता है ढाल का उपयोग करता है.

Abstract

Synaptoneurosomes (SNS) और माउस मस्तिष्क प्रांतस्था के homogenization fractionation के बाद प्राप्त कर रहे हैं. वे vesicles या पृथक टर्मिनलों कि अक्षतंतु टर्मिनलों से दूर तोड़ने जब cortical ऊतक homogenized है resealed हैं. SNS – पूर्व और postsynaptic विशेषताओं को बनाए रखने, जो उन्हें synaptic प्रसारण के अध्ययन में उपयोगी बनाता है. वे आणविक neuronal संकेतन में इस्तेमाल किया मशीनरी बनाए रखने और तेज, भंडारण, और न्यूरोट्रांसमीटर की रिहाई के लिए सक्षम हैं.

उत्पादन और सक्रिय SNS के अलगाव Ficoll, जो साइटोटोक्सिक हो सकता है और उच्च घनत्व, और निस्पंदन और centrifugation तरीकों, जो कम गतिविधि में SNS के यांत्रिक क्षति के कारण परिणाम कर सकते हैं करने के लिए विस्तारित कारण centrifugation की आवश्यकता कर सकते हैं जैसे समस्याग्रस्त medias का उपयोग किया जा सकता है. हालांकि, असंतत Percoll sucrose gradients घनत्व का उपयोग करने के लिए SNS अलग translationally सक्रिय SNS की अच्छी पैदावार के उत्पादन के लिए एक तेजी से विधि प्रदान करता है. ढाल Percoll sucrose विधि त्वरित और कोमल के रूप में इसे isotonic शर्तों को रोजगार, कम और कम centrifugation spins है और centrifugation कदम से बचा जाता है कि गोली SNS और यांत्रिक क्षति का कारण है.

Protocol

1. 1-4 तैयारियां ढाल माध्यम से 500 एमएल बफर (जीएम) एक 2.5 एम sucrose के घोल का 50 एमएल मिश्रण से तैयार करने के लिए, एक 1M Tris – एचसीएल, pH7.5 शेयर, और एक 0.5 मीटर EDTA की 0.1 एमएल की 2.5 एमएल, पीएच 8.0 के साथ शेयर milli- क्यू मात्रा करने के ल…

Discussion

असंतत ढाल Percoll sucrose के साथ साथ वर्णित तैयारी एक त्वरित, विश्वसनीय विधि सक्रिय SNS, जो synaptic प्रसारण प्रयोगों की एक किस्म में इस्तेमाल किया जा सकता है अलग है. इस ढाल विधि है, जो Dunkley एट अल. 3,4 द्वारा विकसित की पद्ध…

Disclosures

The authors have nothing to disclose.

Acknowledgements

हम इलेक्ट्रॉन माइक्रोस्कोपी के लिए मैडिसन विस्कॉन्सिन इलेक्ट्रॉन माइक्रोस्कोप सुविधा विश्वविद्यालय से बीके अगस्त को धन्यवाद देना चाहूंगा. इस काम एनआईएच R01-DA026067 और p30-HD03352 (JSM के लिए) अनुदान द्वारा समर्थित किया गया.

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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