JCGGDB TOP GlycoScience Protocol Online Database

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

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

Authors：
Introduction Protocol References Credit lines
Category
N-Glycans
Protocol Name

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

Authors
 Fujita, Kiyotaka
Faculty of Agriculture, Kagoshima University

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

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

  • Recombinant enzyme (expressed in Candida boidinii) is commercially available from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).
●

5X Reaction buffer : 500 mM sodium acetate buffer (pH 6.0)

●

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
●

Reaction incubator or water bath (100°C, 37°C)
●

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.

 Comment 1
2) 

 Incubate at 30°C for 20 h.

 Comment 0
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.

 Comment 0
4) 

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

 Comment 0
Figure & Legends

Figure & Legends

Fig. 1. Specificity of Endo-M.

Copyrights Creative Commons License   Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
Date of registration：2014-05-28 09:19:32
©2010 - 2023 Ritsumeikan University, AIST & JCGGDB. All Rights Reserved