Subcellular Fractionation of Mouse Brain Homogenates
小鼠脑组织匀浆的亚细胞分离
发布: 2013年08月05日第3卷第15期 DOI: 10.21769/BioProtoc.839 浏览次数: 13969
评审: Xuecai Ge
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参见作者原研究论文
The authors used this protocol in:
Jan 2013
Abstract
This subcellular fractionation protocol is used for separation of cellular organelles based on their density. We have designed and optimized the protocol for separation of subcellular compartments of brain homogenates with focus on the localization and trafficking of transmembrane proteins, but we have also successfully used this protocol for fractionation of other types of tissue. The protocol has two major steps 1) preparation of homogenate from dissected tissue and 2) separation of organelles by centrifugation of homogenates using a continuous sucrose gradient.
Materials and Reagents
- Mice
- PBS
- Phosphatase inhibitor (e.g. PhosStop) (F. Hoffmann-La Roche, catalog number: 04906845001 )
- HEPES
- EDTA
- Distilled water
- Glucose
- Protease inhibitors (e.g. Complete) (F. Hoffmann-La Roche, catalog number: 11697498001 )
- Solution A (see Recipes)
- 2.5 M sucrose stock (see Recipes)
- Sucrose solution (0.8 M and 1.6 M) (see Recipes)
Equipment
- A pair of scissor
- A brain scooper
- Falcon tubes (15 ml)
- Centrifuge
- Ultracentrifuge
- Ultracentrifuge rotor (T70.1 Ti and Sw41Ti )
- Ultracentrifuge tubes for T70.1 Ti rotor (Polycarbonate, 16 x 76 mm) (Beckman Coulter, catalog number: 355651 )
- Ultracentrifuge tubes for SW41 Ti rotor (14 x 89 mm) (Seton Open-Top Polyclear centrifuge tubes, catalog number: 7030 )
- Thomas® Teflon Pestle (A.H Thomas Co., model: 3431-E20 )
- 1 and 10 ml syringes
- Needles (18 and 27 G)
- Gradient Master 107 ip (BioComp, model: 107 ) with marker block and cannula
- Refractometer
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Olsen, D. and Gustafsen, C. (2013). Subcellular Fractionation of Mouse Brain Homogenates. Bio-protocol 3(15): e839. DOI: 10.21769/BioProtoc.839.
- Gustafsen, C., Glerup, S., Pallesen, L. T., Olsen, D., Andersen, O. M., Nykjaer, A., Madsen, P. and Petersen, C. M. (2013). Sortilin and SorLA display distinct roles in processing and trafficking of amyloid precursor protein. J Neurosci 33(1): 64-71.
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分类
神经科学 > 细胞机理
细胞生物学 > 细胞器分离 > 分级分离
细胞生物学 > 组织分析 > 组织分离
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