Stem Microsome Preparation and Mannan Synthase Activity Assay
干细胞微粒体的制备和甘露聚糖合成酶活性检测
发布: 2013年05月20日第3卷第10期 DOI: 10.21769/BioProtoc.569 浏览次数: 8642
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参见作者原研究论文
The authors used this protocol in:
Jan 2013
Abstract
Mannans are hemicellulosic polysaccharides and are present in cell walls of all land plants. Mannan polysaccharides are synthesized by two enzymes, mannan synthase (ManS) for backbone (mannan or glucomannan) synthesis and galactomannan galactosyl transferase for side-chain (galactosyl) addition. Here, a method for ManS activity assay using microsomes freshly isolated from Arabidopsis stems is described. This method can be applied to isolation of microsomes from any tissues of Arabidopsis or any other plants.
Keywords: Arabidopsis (拟南芥)
Cell wall (细胞壁)
Microsomes (微粒体)
Galactomannan (半乳甘露聚糖)
Mannan synthase (甘露聚糖合成酶)
Materials and Reagents
- cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail Tablets (Roche, catalog number: 11836170001 )
- GDP-[14C]-Man (9.694 GBq/mmol, 262.0 mCi/mmol, 76.34 μM) (PerkinElmer, catalog number: NEC536050UC )
- Bicinchoninic acid (BCA) Protein Assay Kit (Pierce Antibodies, catalog number: 23225 or 23227 )
- OptiPhase Supermix Cocktail (PerkinElmer, catalog number: 1200-439 )
- GDP-Man (Sigma-Aldrich, catalog number: G-7377 )
- Carob galactomannan (Megazyme International, catalogue number: P-GALML )
- Sucrose
- MgCl2
- Extraction buffer (EB) (see Recipes)
- 4x ManS assay buffer (see Recipes)
- 1 mM GDP-Man (see Recipes)
- 1% carob galactomannan (see Recipes)
Equipment
- Mortar and pestle
- 13 ml Sarstedt tube (Sarstedt, catalog number: 60.540.500 )
- Centrifuge
- 4 ml plastic liquid scintillation counting vial (PerkinElmer, catalog number: 1200-421 )
- 1450 MicroBeta TriLux Microplate Scintillation and Luminescence Counter (PerkinElmer, catalog number: 1450-024 )
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Wang, Y. (2013). Stem Microsome Preparation and Mannan Synthase Activity Assay. Bio-protocol 3(10): e569. DOI: 10.21769/BioProtoc.569.
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分类
生物化学 > 糖类 > 多糖
生物化学 > 蛋白质 > 活性
细胞生物学 > 细胞器分离 > 微粒体
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