Application Features

• Removal of Core 1 and Core 3 O-linked disaccharide glycans from glycoproteins

Unit Definition

One unit 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 (2) 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 Assay
Two fold serial dilutions of O-Glycosidase are added to a reaction mixture of 5 mg of neuraminidase digested fetuin with 1X GlycoBuffer 2. The reaction mix is then incubated at 37°C for 1 hour. O-linked disaccharide carbohydrates are determined by the Morgan and Elson Assay (2).

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

Storage Temperature

-20°C

Storage Conditions

20 mM Tris-HCl
50 mM NaCl
1 mM EDTA
pH 7.5 @ 25°C

Heat Inactivation

65°C for 10 min

Molecular Weight

Apparent: 147000 daltons

Companion Products

• O-Glycosidase & Neuraminidase Bundle
• Endo H
• Endo H_f
• PNGase F
• PNGase F (Glycerol-free)
• α2-3,6,8 Neuraminidase

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.
3. It is necessary to treat glycoproteins concomitantly with Neuraminidase and O-Glycosidase. Neuraminic Acid residues must be removed in order to allow O-Glycosidase to cleave the O-linked disaccharides. A general Neuraminidase (#P0720) or the O-Glycosidase and Neuraminidase bundle (#E0540S)  is recommended.
4. Under denaturing conditions the enzyme activity is increased two-fold. This observation is substrate dependent.

1. Koutsioulis, D., Landry, D. and Guthrie, E.P. (2008). Glycobiology. 18, 799-805.
2. Morgan, W.T.J. and Elson, L.A. (1994). Biochem. J.. 28, 988-995.

1. What are the typical reaction conditions for O-Glycosidase?
2. I tried using O-Glycosidase on my glycoprotein and didn't see removal of the carbohydrate. What could be the problem?
3. How much O-Glycosidase should I use to remove my carbohydrate under native conditions?
4. How do I inhibit O-Glycosidase?
   
   
5. Do detergents inhibit O-Glycosidase?
6. Can Neuraminidase be used together in a digest with PNGase F and O-Glycosidase?
7. Can I double digest PNGase F and O-Glycosidase?
8. What is the difference between PNGase F, Endo H and O-Glycosidase?
9. Why have the NEB Glycosidase enzymes changed reaction buffers? What are the new reaction buffers and can I still use an enzyme with its old buffer? Where can I find the composition of the old buffers?
10. What are glycosidases and their uses?
11. Is it necessary to treat my glycoprotein concomitantly with Neuraminidase and O-Glycosidase?

1. O-Glycosidase Application Note 1 (P0733)
2. O-Glycosidase (P0733)
3. Endo-α-N-Acetylgalactosaminidase Application Note 1

• Endoglycosidase Selection Chart

• Using Glycosidases to Remove Trim or Modify Glycans on Therapeutic Protein

• NEBioCalculator

• Enzymatic deglycosylation of a protein containing Core 1 O-glycans with O-Glycosidase
• Analysis of a Fusion Protein using the Protein Deglycosylation Mix II and Mass Spectrometry

• Glycoproteomics Brochure
• Glycoproteomics Technical Guide

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

• P0733S_L_v1

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality control's for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

• P0733S_L_v1_0031603
• P0733S_L_v1_0031612
• P0733S_L_v1_0031706
• P0733S_L_v1_0031801

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

• O-Glycosidase
• Glycoprotein Denaturing Buffer
• NP-40
• GlycoBuffer 2

This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).

While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.

For more information about commercial rights, please contact NEB's Global Business Development team at gbd@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.