[14C] Glucose Cell Wall Incorporation Assay for the Estimation of Cellulose Biosynthesis
采用14C 葡萄糖细胞壁掺入实验估测纤维素的生物合成
发布: 2015年09月20日第5卷第18期 DOI: 10.21769/BioProtoc.1589 浏览次数: 7520
评审: Renate WeizbauerAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Nov 2014
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Abstract
Cellulose is synthesized by Cellulose Synthase A proteins at the plasma membrane using the substrate UDP glucose. Herein, we provide a detailed method for measuring the incorporation of radiolabeled glucose into the cellulose fraction of the cell wall. In this method Arabidopsis seedlings are treated for 2 h with a cellulose biosynthesis inhibitor in the presence of radiolabeled glucose, and are subsequently boiled in acetic-nitric acid to solubilize non-cellulosic material. The radiolabeled glucose detected in the insoluble fraction indicates the amount of cellulose synthesized during the experimental timeframe. The short-term nature of this method is a useful tool in determining if inhibition of cellulose biosynthesis is the herbicides primary mode of action.
Materials and Reagents
- Arabidopsis seed (Arabidopsis thaliana L.)
- Dextrose (Sigma-Aldrich, catalog number: D9434 )
- Radiolabeled glucose [glucose D-14C(U)] (American Radiolabeled Chemicals, catalog number: ARC 0122D )
- Acetic Acid, Glacial (Certified ACS) (Fisher Scientific, catalog number: A38-212 )
- Nitric Acid, Fuming (Certified ACS) (Fisher Scientific, catalog number: A202-212 )
- Counting cocktail (Biodegradable Scintillation Cocktails) (Biosafe II, catalog number: 111195 )
- Deionized sterile water (autoclaved at 120 °C for 30 min)
- Murashige & Skoog (MS) Basal Salt Mixture (PhytoTechnology Laboratories®, catalog number: M524 )
- MES monohydrate (Sigma-Aldrich, catalog number: 69889 )
- Model A307 Sample Oxidizer (PerkinElmer, catalog number: A030700 )
- Aluminum foil
- Eppendorf tubes (1.5 ml) (autoclaved at 120 °C for 30 min)
- MS media (see Recipes)
- Acetic-nitric acid reagent (see Recipes)
Equipment
- Eppendorf tube holders
- Pasteur pipets and tips
- 1 L Erlenmeyer flask (autoclaved at 120 °C for 30 min)
- Microscale
- Orbital shaker
- Microcentrifuge
- Glass scintillation vials (20 ml)
- Scintillation Counter (Packard Tri-Carb Liquid Scintillation Counter)
- Autoclave
Procedure
文章信息
版权信息
© 2015 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:
- Brabham, C., Stork, J. and Debolt, S. (2015). [14C] Glucose Cell Wall Incorporation Assay for the Estimation of Cellulose Biosynthesis. Bio-protocol 5(18): e1589. DOI: 10.21769/BioProtoc.1589.
- Brabham, C., Lei, L., Gu, Y., Stork, J., Barrett, M. and DeBolt, S. (2014). Indaziflam herbicidal action: a potent cellulose biosynthesis inhibitor. Plant Physiol 166(3): 1177-1185.
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分类
植物科学 > 植物生物化学 > 糖类
植物科学 > 植物生理学 > 组织分析 > 细胞壁
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