Preparation of Crude Synaptosomal Fractions from Mouse Brains and Spinal Cords
利用小鼠脑和脊髓粗制突触体组分
发布: 2017年08月05日第7卷第15期 DOI: 10.21769/BioProtoc.2423 浏览次数: 14284
评审: Fanny EhretAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jan 2017
Abstract
The current protocol describes the preparation of crude synaptosomal fractions from mouse brain or spinal cord samples. In detail, a sequential protocol yielding crude synaptosomal and light membrane fractions is provided. This fast and easy method might be sufficient to assess the amount of synaptic proteins in down-steam applications like Western-blot or ELISA in e.g., mouse models of Alzheimer’s disease or other neurodegenerative conditions.
Keywords: Brain (脑)
Synaptosome (突触体)
Fractionation (分离)
Homogenization (匀化)
Extraction (萃取)
Synapse (突触)
Background
Analyzing synaptosomes, representing isolated synaptic terminals from neurons, can yield valuable information on synaptic integrity in diverse neurological diseases. They contain membrane-bound compartments that detach from axon terminals after brain homogenization under certain conditions. The current protocol describes a fast and easy method for the enrichment of crude synaptosomal fractions (see Figure 1). These can be either used for quantification of synaptic proteins by Western-blot or can be further purified using density gradient centrifugation to yield highly purified synaptosome subfractions (Gurd et al., 1974). The preparation of crude synaptosomal fractions might be sufficient to assess e.g., the amount of pre- and post-synaptic proteins like SNAP25 or post-synaptic density protein 95 (PSD95) in e.g., mouse models with a neurodegenerative phenotype (Breyhan et al., 2009; Saul and Wirths, 2017). 
Figure 1. Flow-chart describing the sequential extraction procedure
Materials and Reagents
- Pipette tips
10 µl pipette tips (SARSTEDT, catalog number: 70.1130 )
200 µl pipette tips (SARSTEDT, catalog number: 70.760.002 )
1,000 µl pipette tips (SARSTEDT, catalog number: 70.762 )
- Reaction tubes
1.5 ml tubes (SARSTEDT, catalog number: 72.706 )
2 ml tubes (SARSTEDT, catalog number: 72.691 )
- Mice (Protocol has been tested with male and female C57Bl/6 mice of 8-52 weeks)
- Sucrose (≥ 99.5%) (Sigma-Aldrich, catalog number: S0389 )
- HEPES (≥ 99.5%) (Sigma-Aldrich, catalog number: H3375 )
- Protease inhibitor cocktail (cOmpleteTM Mini EDTA-free EasyPack) (Roche Diagnostics, catalog number: 04693159001 )
- Phosphatase inhibitor cocktail 3 (Sigma-Aldrich, catalog number: P0044 )
- Sodium chloride (NaCl) (≥ 99.5%) (Carl Roth, catalog number: 3957 )
- Potassium chloride (KCl) (≥ 99%) (Carl Roth, catalog number: P017 )
- Di-Sodium hydrogen phosphate (Na2HPO4) (≥ 98%) (Carl Roth, catalog number: P030 )
- Potassium di-hydrogen phosphate (KH2PO4) (≥ 99%) (Carl Roth, catalog number: 3904 )
- Lysis buffer (pH 7.5) (see Recipes)
- Phosphate-buffered saline (PBS) (see Recipes)
Equipment
- Pipettes (Research 0.5-10 µl, 10-100 µl, 100-1,000 µl) (Eppendorf)
- Heraeus Biofuge Stratos (Rotor) (Heraeus Holding, catalog number: 3332 )
- Ultrasound sonicator (Emerson Electric, BRANSON, model: 150 )
- Stirring device (Ingenieurbüro CAT M. Zipperer, model: R 50D )
- Glass homogenizer (2 ml) (Carl Roth, catalog number: TT57.1 )
- Teflon pestle (Carl Roth, catalog number: TT63.1 )
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Wirths, O. (2017). Preparation of Crude Synaptosomal Fractions from Mouse Brains and Spinal Cords. Bio-protocol 7(15): e2423. DOI: 10.21769/BioProtoc.2423.
Download Citation in RIS Format
分类
神经科学 > 细胞机理 > 突触生理学
分子生物学 > 蛋白质 > 表达
分子生物学 > 蛋白质 > 蛋白质-蛋白质相互作用
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