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Endo-β-N-acetylglucosaminidase M digestion (Endo-M)

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Introduction Protocol References Credit lines
Category
N-Glycans
Protocol Name

Endo-β-N-acetylglucosaminidase M digestion (Endo-M)

Authors
 Kiyotaka Fujita
Faculty of Agriculture, Kagoshima University

Kenji Yamamoto *
Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University
*To whom correspondence should be addressed.
Keywords

asparagine-linked glycan

endo-β-N-acetylglucosaminidase

N-glycan

endoglycosidase

bi-antennary complex type oligosaccharide

Reagents
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Endo-β-N-acetylglucosaminidase M (Endo-M) from Mucor hiemalis.

  • Recombinant enzyme (expressed in Candida boidinii) is commercially available from Tokyo Chemical Industry (Tokyo, Japan).
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5X Reaction buffer : 500 mM sodium acetate buffer (pH 6.0)

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2X SDS-PAGE sample buffer: 0.125 M Tris-HCl buffer (pH 6.8), 10% β-ME, 4% SDS, 10% sucrose, 0.004% Bromophenol blue

Instruments
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Reaction incubator or water bath (100°C, 37 °C)
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SDS-PAGE system
Methods
1.

Release of oligosaccharides from glycoproteins by using Endo-M.

1) 

 Transfer 20 μL of glycoprotein sample (10 μg/μL), 10 μL of 5X reaction buffer, 2.5 μL of 10 U/mL Endo-M, and 17.5 μL of deionized water into a microtube.

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2) 

 Incubate at 30°C for 20 h.

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3) 

 To examine the release of oligosaccharide, mix 5 μL of the reaction sample and 5 μL of 2X SDS-PAGE sample buffer, and then heat at 100°C for 3 min.

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4) 

 Load 10 µL of the sample mixture on SDS-PAGE gel and run the electrophoresis. Perform either Coomassie blue staining or silver staining.

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Figure & Legends

Figure & Legends

Fig. 1. Specificity of Endo-M.

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Date of registration：2011-09-05 15:48:34
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