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

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 F1 (Endoglycosidase F1 or Endo-F1) from Elizabethkingia meningosepticum (formerly known as Chryseobacterium meningosepticum or Flavobacterium meningosepticum). Recombinant enzymes (expressed in E. coli) are commercially available from Sigma-Aldrich (St. Louis, MO), QA-Bio (San Mateo, CA), and Merck KGaA (Darmstadt, Germany).
●	Endo-β-N-acetylglucosaminidase F2 (Endoglycosidase F2 or Endo-F2) from Elizabethkingia meningosepticum. Recombinant enzymes (expressed in E. coli) are commercially available from Sigma-Aldrich, QA-Bio, AMS Biotechnology (Witney, UK), Prozyme Inc. (San Leandro, CA), and Merck KGaA.
●	Endo-β-N-acetylglucosaminidase F3 (Endoglycosidase F3 or Endo-F3) from Elizabethkingia meningosepticum. Recombinant enzymes (expressed in E. coli) are commercially available from Sigma-Aldrich, QA-Bio, AMS Biotechnology, and Merck KGaA.
●	5X Reaction buffer for Endo-F1: 250 mM sodium phosphate buffer (pH5.5)
●	5X Reaction buffer for Endo-F2 and Endo-F3: 250 mM sodium acetate buffer (pH4.5)
●	Denaturation solution: 2% w/v sodium lauryl sulfate (SDS), 1 M β-mercaptoethanol (β-ME)
●	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 (Merck Millipore, Billerica, MA)

Methods

Release of oligosaccharides from glycoproteins by using Endo-F1, -F2, or -F3

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-F1, Endo-F2, or Endo-F3 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-F1, -F2, or -F3

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-F1, Endo-F2, or Endo-F3 solution to attain a final enzyme concentration of 0.5–10 U /mL.	Comment 0

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-F1, -F2, and -F3.

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Date of registration：2013-11-15 16:44:58