Analysis of Starch Synthase Activities in Wheat Grains using Native-PAGE
采用非变性聚丙烯酰胺凝胶电泳分析麦粒中淀粉合成酶的活性
发布: 2016年01月20日第6卷第2期 DOI: 10.21769/BioProtoc.1713 浏览次数: 10730
评审: Samik BhattacharyaSaminathan ThangasamyCindy Ast
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参见作者原研究论文
The authors used this protocol in:
May 2014
Abstract
Starch synthases are one class of key enzymes involving in the synthesis of cereal starch, which transfer glucose from ADP-glucose to the non-reducing end of pre-existing α-(1-4)-liked glucosyl chains of amylopectin. This protocol is highly reproducible for assaying activities for starch synthase I and IIIa in wheat and barley endosperm at qualitative level and quantitative level. The protocol includes separating proteins isolated from developing endosperm with native-PAGE containing glycogen from oyster, incubating protein gels with ADP-glucose solution, and staining gels with iodine solution. The method allows researchers to compare the levels or changes of starch synthase activities.
Keywords: Starch synthase (淀粉合成酶)
Enzymatic activity assay (酶活性测定)
Wheat (小麦)
Grain (粮食)
Native-PAGE (本地页面)
Materials and Reagents
- Glad-wrap (Microwave safe, Capri)
- Kimwipe (Kimtech Science* Kimwipes delicate task wipers) (Kimberly-Clark, catalog number: 34133 )
- Gel-cassette (size of 100 mm x 100 mm with 1 mm gap) (Invitrogen, catalog number: NC2010 )
Note: Currently, it is “Thermo Fisher Scientific, Novex™, catalog number: NC2010”. - 15 ml blue cap Falcon tube (sterile) (Thermo Fisher Scientific)
- Epipestle (bioWORLD, catalog number: 42741000-1 )
- 96 well UV microplate (Flat bottom) (Thermo Fisher Scientific, catalog number: 8404 )
- Cuvette (plastic) (SARSTEDT AG & Co, catalog number: 67.746 )
- MilliQ water
- 70% ethanol (Chem Supply, catalog number: 64-17-5 )
- 2% agarose (Progen, catalog number: 200-0011 )
- Tris (2-Amino-2-hydroxymethyl-propane-1, 3-diol) (VWR International, catalog number: 103157P )
- Glycogen from oyster (Sigma-Aldrich, catalog number: G8751 )
- Acrylamide (40% Acrylamide/Bis Solution, 37.5:1) (Bio-Rad Laboratories, catalog number: 161-0148 )
- TEMED (Tetramethylethylenediamine) (AMRESCO, catalog number: 0761-25 ML )
- APS (Ammonia persulfate) (Sigma-Aldrich, catalog number: A-7460 )
- Coomassie Plus Protein Assay Reagent (Thermo Fisher Scientific, catalog number: 1856210)
- Bovine serum albumin (BSA) (Freeze dried, Reagent Grade, pH 7) (Moregate Biotech)
- HCl (Ajax Finechem Pty, catalog number: 1367-2.5 L )
- Ammonium Sulfate [(NH4)2SO4] (AR) (Chem Supply, catalog number: AA014-500 G )
- Magnesium Chloride Hexahydrate (MgCl2) (AR) (Chem Supply, catalog number: MA029-500 G )
- β-mercaptoethanol (Sigma-Aldrich, catalog number: M-7154 )
- Adenosine-5’-diphosphoglucose disodium salt (ADPG) (Sigma-Aldrich, catalog number: A0627-250 mg )
- Protease inhibitor cocktail for plant cell and tissue extracts (Sigma-Aldrich, catalog number: P9599 )
- DL-Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D-9779 )
- Glycine (Chem Supply, catalog number: GA007-500 G )
- Bromophenol blue (Sigma-Aldrich, catalog number: B0126-25 G )
- Glycerol (AR) (Chem Supply, catalog number: GA010-2.5 L )
- Extraction buffer (see Recipes)
- 1x electrophoresis buffer (see Recipes)
Equipment
- Gel tank (XCell SureLock) (Invitrogen, catalog number: EI0001 )
Note: Currently, it is “Thermo Fisher Scientific, NovexTM, catalog number: EI0001”. - Pipette (Thermo Fisher Scientific, model: Finnpipette F1 )
- Spectrophotometer (Agilent Technologies, model: Cary 300 Bio UV-Visible Spectrophotometer )
- Microplate reader (BMG LABTECH GmbH, model: FLUOstar Omega Microplate Reader )
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Li, Z. (2016). Analysis of Starch Synthase Activities in Wheat Grains using Native-PAGE. Bio-protocol 6(2): e1713. DOI: 10.21769/BioProtoc.1713.
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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 > 营养
生物化学 > 糖类 > 多糖
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