Purification of Starch Granules from Arabidopsis Leaves and Determination of Granule-Bound Starch Synthase Activity
植物叶片淀粉粒的纯化和淀粉粒结合的淀粉合酶的活性分析
发布: 2014年12月05日第4卷第23期 DOI: 10.21769/BioProtoc.1316 浏览次数: 9455
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参见作者原研究论文
The authors used this protocol in:
Feb 2014
Abstract
Starch constitutes the most important carbon reserve in plants and is composed of branched amylopectin and linear amylose. The latter is synthesized exclusively by the Granule-Bound Starch Synthase (GBSS, EC 2.4.1.21). Here we report a readily reproducible, specific and highly sensitive protocol, which includes the isolation of intact starch granules from Arabidopsis thaliana leaves and the subsequent determination of GBSS activity. We have applied this method to study GBSS activity in diurnal cycles in vegetative growth and during the photoperiodic transition to flowering in Arabidopsis (Tenorio et al., 2003; Ortiz-Marchena et al., 2014).
Keywords: Starch granule (淀粉颗粒)
Starch purification (淀粉净化)
GBSS activity (GBSS活性)
Granule-bound starch synthase (颗粒结合型淀粉合酶)
Materials and Reagents
- Plant materials
Note: Arabidopsis thaliana (A. thaliana) were grown in controlled cabinets on peat-based compost. - Liquid N2
- HEPES (Sigma-Aldrich, catalog number: H4034 )
- Potassium hydroxide pellets (Panreac Applichem, catalog number: A0566 )
- TritonTM X-100 (Sigma-Aldrich, catalog number: X100 )
- Miracloth (Merck KGaA, catalog number: 475855 )
- Percoll® (Sigma-Aldrich, catalog number: P1644 )
- Glycogen (from rabbit liver Type III) (Sigma-Aldrich, catalog number: G8876 )
- Tricine (Sigma-Aldrich, catalog number: T0377 )
- Potassium acetate (Sigma-Aldrich, catalog number: P1147 )
- DL-Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
- Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) (Sigma-Aldrich, catalog number: E5134 )
- Sodium hydroxide pellets (Panreac Applichem, catalog number: 131687 )
- Maltotriose (Sigma-Aldrich, catalog number: M8378 )
- Adenosine 5’ diphospho, [D-glucose-14C(U)] ammonium salt (ADP [U-14C] glucose) (American Radiolabeled Chemicals, catalog number: ARC 3297 )
- Methanol (CARLO ERBA Reagents, catalog number: 412532 )
- Potassium chloride (AppliChem GmbH, catalog number: A2939 )
- Ecolite(+)TM liquid scintillation cocktail (MP Biomedicals, catalog number: 882475 )
- 1 M HEPES-KOH (see Recipes)
- 1 M Tricine (see Recipes)
- 0.25 M Potassium acetate (see Recipes)
- 1 M DTT (see Recipes)
- 0.5 M EDTA (see Recipes)
- 0.1 M Maltotriose (see Recipes)
- 3 M KCl (see Recipes)
- 20 mg/ml Glycogen solution (see Recipes)
- Extraction buffer (see Recipes)
- Percoll buffer (see Recipes)
- Washing buffer (see Recipes)
- Precipitation buffer (see Recipes)
Equipment
- Small mortar and pestle
- 1.5 ml microfuge tubes
- Automatic pipettes
- Precision scale
- Centrifuge (Eppendorf, model: 5810 R )
- Microcentrifuge (Eppendorf, model: 5424 )
- Scintillation Counter (Beckman Coulter, model: LS 6000 IC )
- Scintillation vials (Sigma-Aldrich, catalog number: Z376817 )
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Albi, T., Ortiz-Marchena, M. I., Ruiz, M. T., Romero, J. M. and Valverde, F. (2014). Purification of Starch Granules from Arabidopsis Leaves and Determination of Granule-Bound Starch Synthase Activity. Bio-protocol 4(23): e1316. DOI: 10.21769/BioProtoc.1316.
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分类
植物科学 > 植物新陈代谢 > 糖类
植物科学 > 植物生物化学 > 蛋白质
生物化学 > 糖类 > 多糖
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