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

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.

1. 1-4 तैयारियां <ol><li> 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...

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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...

<table border="1"><tbody><tr><th> अभिकर्मक के नाम </th><th> कंपनी </th><th> सूचीपत्र संख्या </th><th> टिप्पणियां (वैकल्पिक) </th></tr><tr><td> माइक्रो बीसीए प्रोटीन परख किट </td><td> पियर्स </td><td> 23235 </td><td></td></tr><tr><td> 2 CaCl </td><td> मछुआ </td><td> C79-500 </td><td></td></tr><tr><td> सीओ 2 गैस </td><td> Airgas (UW-एमडीएस) </td><td> 50 सीडी </td><td></td></tr><tr><td> EDTA </td><td> आरपीआइ </td><td> E57020 </td><td></td></tr><tr><td> EtOH </td><td> मछुआ </td><td> A407SK - 4 </td><td></td></tr><tr><td> एचसीएल </td><td> मछुआ </td><td> A142-212 </td><td></td></tr><tr><td> Percoll </td><td> जीई हेल्थकेयर </td><td> 17-0891-01 </td><td></td></tr><tr><td> 2 के.एच. पीओ 4 </td><td> मछुआ </td><td> P285-500 </td><td></td></tr><tr><td> PierceSDS पृष्ठ नमूना तैयारी किट </td><td> पियर्स </td><td> 89888 </td><td></td></tr><tr><td> NaCl </td><td> आरपीआइ </td><td> S23020 </td><td></td></tr><tr><td> 3 NaHCO </td><td> मछुआ </td><td> BP328 - 500 </td><td></td></tr><tr><td> ना दो चार फोटो </td><td> मछुआ </td><td> S381 - 500 </td><td></td></tr><tr><td> इक्षुसिता </td><td> आरपीआइ </td><td> S24060 </td><td></td></tr><tr><td> Tris बेस </td><td> आरपीआइ </td><td> T60040 </td><td></td></tr><tr><td> Tetrodotoxin </td><td> सिग्मा </td><td> T5651 </td><td></td></tr><tr><td> एक्सप्रेस प्रो लेबल S35 आसान टैग मिक्स </td><td> Perkin एल्मर </td><td> NEG772 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><th> उपकरण </th><th> कंपनी </th><th> सूचीपत्र संख्या </th><th> टिप्पणियां (वैकल्पिक) </th></tr><tr><td> विच्छेदन उपकरण </td><td></td><td></td><td></td></tr><tr><td> Dounce homogenizer, 7 एमएल (दो गिलास के साथ आता है लेबल &quot;A&quot; और &quot;बी&quot; pestles) </td><td> व्हिटन </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 अपकेंद्रित्र </td><td> Beckman कल्टर </td><td> 366830 </td><td></td></tr><tr><td> GH 3.8 रोटर, झूलते बाल्टी रोटर </td><td> Beckman कल्टर </td><td> 360581 </td><td></td></tr><tr><td> Beckman अपकेंद्रित्र J2-21 </td><td> Beckman </td><td></td><td></td></tr><tr><td> टोपी के साथ Beckman ट्यूबों </td><td> Beckman </td><td> 355672 </td><td></td></tr><tr><td> सफेद दीवारों एडेप्टर </td><td> Beckman </td><td> 342327 </td><td></td></tr><tr><td> नीले घिरी एडेप्टर </td><td> Beckman </td><td></td><td></td></tr><tr><td> जावेद-17 रोटर, रोटर निश्चित कोण </td><td> Beckman </td><td> 369691 </td><td></td></tr></tbody></table> पश्चिमी blots के लिए इस्तेमाल किया एंटीबॉडी की तालिका: <table border="1"><tbody><tr><th> एंटीबॉडी के नाम </th><th> कंपनी </th><th> सूचीपत्र संख्या </th><th> होस्ट प्रजाति </th><th> मन्दन फैक्टर </th></tr><tr><td> β-actin </td><td> सिग्मा </td><td> A5441 </td><td> माउस </td><td> 1:2000 </td></tr><tr><td> GFAP </td><td> सांता क्रुज़ </td><td> SC - 65,343 </td><td> माउस </td><td> 1:200 </td></tr><tr><td> GP73 </td><td> सांता क्रुज़ </td><td> SC-+१३४५०९ </td><td> खरगोश </td><td> 1:200 </td></tr><tr><td> HSC70 </td><td> सांता क्रुज़ </td><td> SC-7298 </td><td> माउस </td><td> 1:200 </td></tr><tr><td> Laminβ </td><td> सांता क्रुज़ </td><td> SC-६,२६१ </td><td> बकरी </td><td> 1:200 </td></tr><tr><td> Prohibitin </td><td> सांता क्रुज़ </td><td> SC - 28,259 </td><td> खरगोश </td><td> 1:200 </td></tr><tr><td> PSD95 </td><td> Millipore </td><td> MAB1596 </td><td> माउस </td><td> 5 μg / μL </td></tr><tr><td> SNAP25 </td><td> AbCam </td><td> ab5666 - 100 </td><td> खरगोश </td><td> 1:2000 </td></tr><tr><td> Synaptophysin </td><td> Millipore </td><td> MAB368 </td><td> माउस </td><td> 1:500 </td></tr><tr><td> β-3-ट्यूबिलिन </td><td> सांता क्रुज़ </td><td> SC - 80,016 </td><td> माउस </td><td> 1:200 </td></tr></tbody></table>

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

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.

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

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.

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

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

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