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

Authors：

Category

N-Glycans

Protocol Name

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

●	Endoglycosidase D from Streptococcus pneumoniae (formerly known as Diplococcus pneumoniae). Enzymes purified from culture filtrate are commercially available from Seikagaku Biobusiness Corp. (Tokyo, Japan) and United States Biological (Swampscott, MA).
●	Neuraminidase, β-galactosidase, and β-N-acetylglucosaminidase are commercially available from Sigma-Aldrich (St. Louis, MO) and others.
●	5× Reaction buffer : 500 mM citrate phosphate buffer (pH 6.0)
●	2× 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 (37°C)
●	SDS-PAGE system

Methods

Release of oligosaccharides from glycoproteins by using Endo-D.

1)	Transfer 20 μL of glycoprotein sample (10 μg/μL), 10 μL of 5× reaction buffer, 2.5 μL of 400 mU Endo-D, and 10 μL of deionized water into a microtube. If complete removal of oligosaccharides is required, 2.5 μL of 800 mU neuraminidase, 2.5 μL of 400 mU β-galactosidase, and 2.5 μL of 400 mU β-N-acetylglucosaminidase should be added with Endo-D.	Comment 1

2)	Incubate at 37°C for 20 h.	Comment 0

3)	To examine the release of oligosaccharide, mix 5 μL of the reaction sample and 5 μL of 2× SDS-PAGE sample buffer, and then heat at 100°C for 3 min.	Comment 0

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

Fig. 1. Specificity of Endo-D.

Copyrights

Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses

Date of registration：2016-01-21 11:34:01