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

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

●	Endo-β-N-acetylglucosaminidase H (Endoglycosidase H or Endo-H) from Streptomyces plicatus. Enzymes purified from culture filtrate are commercially available from Sigma-Aldrich (St. Louis, MO), Seikagaku Biobusiness Co. (Tokyo, Japan), AMS Biotechnology (Witney, UK) and United States Biological (Swampscott, MA). Recombinant enzymes (expressed in E. coli) are commercially available from Roche Diagnostics GmbH (Mannheim, Germany), New England Bio Labs Inc. (Billerica, MA), Sigma-Aldrich (St. Louis, MO), QA-Bio (San Mateo, CA), AMS Biotechnology (Witney, UK), Prozyme (San Leandro, CA) and Merck KGaA (Darmstadt, Germany).
●	Denaturation solution: 2% w/v sodium lauryl sulfate (SDS), 1 M β-mercaptoethanol (β-ME)
●	5X Reaction buffer : 250 mM sodium phosphate buffer (pH5.5)
●	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
●	Microcon Ultracel YM-10 (Millipore)

Methods

Release of oligosaccharides from glycoproteins by using Endo-H

1)	Transfer 20 μL of glycoprotein sample (10 μg/μL), 10 μL of 5X Reaction buffer, and 17.5 μL of deionized water into a microtube.	Comment 1

2)	Add 2 μL of Endo-H solution to attain a final enzyme concentration of 0.5~10 U /mL.	Comment 0

3)	Incubate at 37°C for 18 h.	Comment 0

4)	Terminate the reaction by heating at 100°C for 3 min.	Comment 1

Separate the oligosaccharides from the deglycosylated protein by ultrafiltration using Microcon Ultracel YM-10 (10-kDa cut-off membrane; Millipore).

Comment 1

SDS-PAGE analysis of glycoproteins deglycosylated with Endo-H.

1)	Transfer 20 μL of glycoprotein sample (10 μg/μL), 10 μL of 5X reaction buffer, 2.5 μL of denaturation solution, and 15.5 μL of deionized water into a microtube.	Comment 1

2)	Heat at 100°C for 3 min.	Comment 0

3)	Add 2 μL of Endo-H solution to attain a final enzyme concentration of 0.5~10 U /mL.	Comment 1

4)	Incubate at 37°C for 18 h.	Comment 0

5)	Add 50 μL of 2X SDS-PAGE sample buffer, and then heat at 100°C for 3 min.	Comment 0

Load 10 µL of the sample 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-H.

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Date of registration：2013-11-21 18:29:12