Determination of D-galactofuranose Content of Galactomannoproteins in Aspergillus nidulans
构巢曲霉中半乳甘露糖蛋白的D-呋喃半乳糖含量测定
发布: 2014年09月05日第4卷第17期 DOI: 10.21769/BioProtoc.1223 浏览次数: 8228
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参见作者原研究论文
The authors used this protocol in:
Dec 2013
Abstract
Galactofuranose (Galf) is a component of several polysaccharides and glycoconjugates in certain species of filamentous fungi. Galf residues are frequently found in Aspergillus glycoproteins, including N-glycans and O-mannose glycans that modify many cell wall proteins and extracellular enzymes. It is known that furanoses, contained in oligosaccharides, are detected as pyranoses after hydrolysis, and that D-galactopyranose is not contained in the galactomannoproteins of Aspergillus spp. To determine the levels of D-galactofuranose in galactomannoproteins extracted from Aspergillus nidulans (A. nidulans), we measured the amount of D-galactopyranose production after galactomannoproteins hydrolysis. The method described in this manuscript allows determination of the D-galactofuranose content of galactomannoproteins in Aspergillus spp.
Keywords: Galactofuranose (呋喃)
Aspergillus (曲霉)
Cell wall (细胞壁)
Galactomannan (半乳甘露聚糖)
Materials and Reagents
- Aspergillus nidulans conidia
- Trisodium citrate dihydrate (C6H5Na3O7.2H2O) (Wako Pure Chemical Industries, catalog number: 191-01785 )
- Citric acid (C6H8O7) (Wako Pure Chemical Industries, catalog number: 030-05525 )
- Ethyl-4-aminobenzoate (C9H11NO2) (ABEE) (Tokyo Chemical Industry, catalog number: A0271 )
- Sodium cyanoborohydride (NaBH3CN) (Tokyo Chemical Industry, catalog number: S0396 )
- Chloroform (Wako Pure Chemical Industries, catalog number: 038-02606 )
- Ethanol (Wako Pure Chemical Industries, catalog number: 050-00446 )
- Trifluoroacetic acid (TFA) (Wako Pure Chemical Industries, catalog number: 206-10731 )
- Glacial acetic acid (Wako Pure Chemical Industries, catalog number: 012-00245 )
- Methanol (Wako Pure Chemical Industries, catalog number: 136-09475 )
- D-galactose (Sigma-Aldrich, catalog number: G0750 )
- ABEE labeling mixture (see Recipes)
- HPLC solvent A (see Recipes)
- HPLC solvent B (see Recipes)
- Minimal medium (see Recipes)
- Hutner's trace elements (see Recipes)
Equipment
- 50-ml plastic centrifuge tube (e.g., Greiner Bio-One GmbH)
- 1.5-ml conical screw cap tube and cap
- Spreader
- 500-ml Sakaguchi flasks
- Centrifuge
- Rotator (e.g., TAITEC)
- High-performance liquid chromatography (HPLC) system equipped with a fluorescence detector (Hitachi, LaChrom Elite, model: L-2485) and software for HPLC peak analysis (e.g., Hitachi, model: D-2000 Elite HPLC)
- Centrifugal evaporator (e.g., SpeedVac®)
- Heat block or water bath
- GlycoScope Honenpak C18 column (4.6 mm x 75 mm) (COSMO BIO, catalog number: JOM-J715-1PC)
- Filter paper (Munktell & Filtrak GmbH, catalog number: 113053 )
- Dialysis membrane (BioDesign Inc. of New York, catalog number: D102 )
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Oka, T., Katafuchi, Y., Fukuda, K., Ekino, K., Goto, M. and Nomura, Y. (2014). Determination of D-galactofuranose Content of Galactomannoproteins in Aspergillus nidulans. Bio-protocol 4(17): e1223. DOI: 10.21769/BioProtoc.1223.
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分类
微生物学 > 微生物生物化学 > 糖类
微生物学 > 微生物新陈代谢 > 糖类
生物化学 > 糖类 > 糖蛋白
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