1 set of this bundle includes 2,000,000 units of O-Glycosidase and 2,000 units of Neuraminidase.

O-Glycosidase,   also known as Endo-α-N-Acetylgalactosaminidase,   catalyzes the removal of

Core 1 and Core 3 O-linked disaccharides from glycoproteins. 

Neuraminidase is the common name for Acetyl-neuraminyl  hydrolase ( Sialidase ).   This Neura-minidase catalyzes the hydrolysis of α2-3, α2-6, α2-8 linked N-acetyl-neuraminic  acid  residues

from glycoproteins and oligosaccharides. 

Product Source

O-Glycosidase is cloned from Enterococcus faecalis and expressed in E.coli (1). Neuraminidase

is cloned from Clostridium perfringens (2) and overexpressed in E. coli (3).

Reagents Supplied

The following reagents are supplied with this product:

Unit Definition

One unit of O-Glycosidase is defined as the amount of enzyme required to remove 0.68 nmol of O-linked disaccharide from 5 mg of Neuraminidase digested, non-denatured fetuin in 1 hour at 37°C in a total reaction volume of 100 µl (1 unit of both O-Glycosidase and PNGase F will remove equivalent molar amounts of O-linked disaccharides and N-linked oligosaccharides, respectively).

Non-denaturing Unit Definition of O-Glycosidase:
Two  fold  serial  dilutions  of  O-Glycosidase  are added to a reaction mixture of 5 mg  of Neura-minidase  digested  fetuin with 1 X GlycoBuffer 2. The  reaction  is  then  incubated at 37°C for 1 hour. O-linked disaccharide carbohydrates are determined by Morgan and Elson Assay (4). Note: Under  denaturing  conditions  the  enzyme  activity  is  increased  two-fold. This observation  is substrate dependent.

Unit Definition of Neuraminidase:
One unit of Neuraminidase is defined as the amount of enzyme required to cleave > 95% of the   terminal α-Neu5Ac from 1 nmol Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc-7-amino-4-methyl-   coumarin (AMC), in 5 minutes at 37°C in a total reaction volume of 10 µl.

1X Glycoprotein Denaturing Buffer
0.5% SDS
40 mM DTT

1X NP-40
1% NP-40 in MilliQ-H₂O

Reaction Conditions

1X GlycoBuffer 2
Incubate at 37°C

1X GlycoBuffer 2:
50 mM sodium phosphate
pH 7.5 @ 25°C

Heat Inactivation

Companion Products

α2-3,6,8 Neuraminidase

O-Glycosidase

PNGase F

PNGase F (Glycerol-free)

Endo H

Endo H_f

1.Since O-Glycosidase is inhibited by SDS, it is essential to have NP-40 in the reaction mixture. It is not known why this non-ionic detergent counteracts the SDS inhibition at the present  time. Double digest with Endo H must have NP-40 present (NP-40 does not inhibit Endo H).

2.To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required.

1.Koutsioulis, D., Landry, D. and Guthrie, E.P. (2008). Glycobiology. 18, 799-805.

2.Roggentin, P. et al. (1988). FEBS Lett. 238, 31-34.

3.Guan, C. unpublished observations. New England Biolabs.

4.Morgan, W.T.J. and Elson, L.A. (1934). Biochem. J. 28, 988-995.

1 set of this bundle includes 2,000,000 units of O-Glycosidase and 2,000 units of Neuraminidase.

O-Glycosidase,   also known as Endo-α-N-Acetylgalactosaminidase,   catalyzes the removal of

Core 1 and Core 3 O-linked disaccharides from glycoproteins. 

Neuraminidase is the common name for Acetyl-neuraminyl  hydrolase ( Sialidase ).   This Neura-minidase catalyzes the hydrolysis of α2-3, α2-6, α2-8 linked N-acetyl-neuraminic  acid  residues

from glycoproteins and oligosaccharides. 

Product Source

O-Glycosidase is cloned from Enterococcus faecalis and expressed in E.coli (1). Neuraminidase

is cloned from Clostridium perfringens (2) and overexpressed in E. coli (3).

Reagents Supplied

The following reagents are supplied with this product:

Unit Definition

One unit of O-Glycosidase is defined as the amount of enzyme required to remove 0.68 nmol of O-linked disaccharide from 5 mg of Neuraminidase digested, non-denatured fetuin in 1 hour at 37°C in a total reaction volume of 100 µl (1 unit of both O-Glycosidase and PNGase F will remove equivalent molar amounts of O-linked disaccharides and N-linked oligosaccharides, respectively).

Non-denaturing Unit Definition of O-Glycosidase:
Two  fold  serial  dilutions  of  O-Glycosidase  are added to a reaction mixture of 5 mg  of Neura-minidase  digested  fetuin with 1 X GlycoBuffer 2. The  reaction  is  then  incubated at 37°C for 1 hour. O-linked disaccharide carbohydrates are determined by Morgan and Elson Assay (4). Note: Under  denaturing  conditions  the  enzyme  activity  is  increased  two-fold. This observation  is substrate dependent.

Unit Definition of Neuraminidase:
One unit of Neuraminidase is defined as the amount of enzyme required to cleave > 95% of the   terminal α-Neu5Ac from 1 nmol Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc-7-amino-4-methyl-   coumarin (AMC), in 5 minutes at 37°C in a total reaction volume of 10 µl.

1X Glycoprotein Denaturing Buffer
0.5% SDS
40 mM DTT

1X NP-40
1% NP-40 in MilliQ-H₂O

Reaction Conditions

1X GlycoBuffer 2
Incubate at 37°C

1X GlycoBuffer 2:
50 mM sodium phosphate
pH 7.5 @ 25°C

Heat Inactivation

 Companion Products

α2-3,6,8 Neuraminidase

O-Glycosidase

PNGase F

PNGase F (Glycerol-free)

Endo H

Endo H_f

1.Since O-Glycosidase is inhibited by SDS, it is essential to have NP-40 in the reaction mixture. It is not known why this non-ionic detergent counteracts the SDS inhibition at the present  time. Double digest with Endo H must have NP-40 present (NP-40 does not inhibit Endo H).

2.To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required.

 1.Koutsioulis, D., Landry, D. and Guthrie, E.P. (2008). Glycobiology. 18, 799-805.

2.Roggentin, P. et al. (1988). FEBS Lett. 238, 31-34.

3.Guan, C. unpublished observations. New England Biolabs.

4.Morgan, W.T.J. and Elson, L.A. (1934). Biochem. J. 28, 988-995.