Product Analysis of Starch Active Enzymes by TLC
采用 TLC法对淀粉活化酶进行成品分析
发布: 2015年10月20日第5卷第20期 DOI: 10.21769/BioProtoc.1621 浏览次数: 13464
评审: Valentine V TrotterLaura Molina-GarcíaAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jan 2015
Abstract
Thin layer chromatography (TLC) is a useful technique for detecting the presence of monosaccharides through to oligosaccharides, though it needs to be optimized for the specific sugars that are analyzed. Here we present a method for visualizing the reaction product(s) of starch active enzymes, which can contain α-1, 4 linked and α-1, 6 linked glucose. This was first published in Molecular Microbiology (Cockburn et al., 2015). The TLC protocol is an adapted version of that published by Robyt and Mukerjea (Robyt and Mukerjea, 1994). For a summary of the products generated by starch active enzymes see the review by Hii et al. (2012).
Keywords: Thin Layer Chromatography (薄层色谱法)
Starch (淀粉)
Amylase (淀粉酶)
Maltooligosaccharides (麦芽寡糖)
Materials and Reagents
- TLC Silica gel 60 F254 20 x 20 cm plates (Note 1) (Merck Millipore Corporation, catalog number: 1.05715.0001 )
- Filter paper circles (15 cm diameter) (VWR International, catalog number: 470204-480 )
- MilliQ quality water (dH2O)
- Acetonitrile (anhydrous) (Sigma-Aldrich, catalog number: 271004 )
- Ethyl acetate (anhydrous) (Sigma-Aldrich, catalog number: 270989 )
- 2-propanol (Sigma-Aldrich, catalog number: I9516 )
- N-(1-naphthyl)ethylenediamine dihydrochloride (Sigma-Aldrich, catalog number: 222488 )
- Methanol (anhydrous) (Sigma-Aldrich, catalog number: 322415 )
- Sulfuric acid (Fluka, catalog number: 84716 )
Note: Currently, it is “ Sigma-Aldrich, catalog number: 84716”. - Dimethyl sulfoxide (DMSO) (Thermo Fisher Scientific, catalog number: D128 )
- α-D-Glucose (anhydrous) (Sigma-Aldrich, catalog number: 158968 )
- Maltose monohydrate (Sigma-Aldrich, catalog number: M9171 )
- Maltotriose (Sigma-Aldrich, catalog number: M8378 )
- Maltotetraose, DP4 (Sigma-Aldrich, catalog number: 47877 )
- Maltopentaose (TCI America, catalog number: M1023 )
- Maltohexaose (Sigma-Aldrich, catalog number: M9153 )
- Maltoheptaose (Sigma-Aldrich, catalog number: 284017 )
- Isomaltose (Sigma-Aldrich, catalog number: I7253 )
- D-Panose (Sigma-Aldrich, catalog number: P2407 )
- Glycogen from bovine liver (Sigma-Aldrich, catalog number: G0885 )
- Amylopectin from potato starch (Sigma-Aldrich, catalog number: A8515 )
- Amylose from potato (Sigma-Aldrich, catalog number: A0512 )
- Dextran from Leuconostoc spp. (Sigma-Aldrich, catalog number: 31389 )
- Pullulan from Aureobasidium pullulans (Sigma-Aldrich, catalog number: P4516 )
- Your enzyme of interest and appropriate buffer
- Mobile phase (see Recipes)
- Staining solution (see Recipes)
- Oligosaccharide standards (see Recipes)
- Glycogen solution (see Recipes)
- Amylopectin solution (see Recipes)
- Amylose solution (see Recipes)
- Dextran solution (see Recipes)
Equipment
Note: All equipment needed is shown in Figure 1.
- TLC developing chamber (VWR International, catalog number: 21432-739 )
- Chromatography sprayer (Note 2) (Sigma-Aldrich, catalog number: Z529729 )
- Oven capable of reaching 120 °C (Boekel Scientific, catalog number: 107905 )
- (Optional) MilwaukeeTM Dual Temperature Heat Gun (Thermo Fisher Scientific, Fisher Scientific, catalog number: 19-313-598 )
- CorningTM ScholarTM PC-170 Hotplate (Thermo Fisher Scientific, Fisher Scientific, catalog number: 13-641-564 )
Figure 1.Equipment needed for this protocol
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Cockburn, D. and Koropatkin, N. (2015). Product Analysis of Starch Active Enzymes by TLC. Bio-protocol 5(20): e1621. DOI: 10.21769/BioProtoc.1621.
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分类
微生物学 > 微生物生物化学 > 蛋白质
生物化学 > 糖类 > 多糖
生物化学 > 蛋白质 > 活性
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