Product Class: Other

O-Glycosidase & α2-3,6,8 Neuraminidase Bundle
NEBU cloned at NEB recombinant 37

Product Introduction

This bundle contains 1 vial each of O-Glycosidase (NEB #P0733S) and a2-3,6,8 Neuraminidase (NEB #P0720S), which can be used simultaneously for the removal of terminal sialic acid residues and core 1 and core 3 O-glycans.

  • Recombinant enzymes with no detectable endoglycosidase or other exoglycosidase contaminating activities
  • Glycerol-free for optimal performance in HPLC and mass spectrometry analysis
  • ≥95% purity, as determined by SDS-PAGE and intact ESI-MS
  • Optimal activity and stability for up to 24 months

Catalog # Size Concentration
E0540S 1.0 set

Product Information

Description

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 Neuraminidase catalyzes the hydrolysis of α2-3, α2-6, and 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).
This product is related to the following categories:
Endoglycosidases Products,
This product can be used in the following applications:
Expression Systems,
Glycan Sequencing,
Protein Digestion,
Proteomics,
Recombinant Glycoprotein Expression,
Glycoprotein Analysis

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • E0540S     -20    
      α2-3,6,8 Neuraminidase P0720SVIAL -20 1 x 0.04 ml 50,000 units/ml
      O-Glycosidase P0733SVIAL -20 1 x 0.05 ml 4.0E7 units/ml
      GlycoBuffer 2 B3704SVIAL -20 1 x 1 ml 10 X
      NP-40 B2704SVIAL -20 1 x 1 ml 10 %
      Glycoprotein Denaturing Buffer B1704SVIAL -20 1 x 1 ml 10 X

Properties & Usage

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 Neuraminidase digested fetuin with 1X 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)

Product Notes

  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.

References

  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.

Protocols, Manuals & Usage

Protocols

  1. O-Glycosidase Application Note 1 (P0733)
  2. O-Glycosidase (P0733)

Usage & Guidelines

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. What are the typical reaction conditions for O-Glycosidase?
  2. Is it necessary to treat my glycoprotein concomitantly with Neuraminidase and O-Glycosidase?
  3. What is the difference between PNGase F, Endo H and O-Glycosidase?
  4. How much O-Glycosidase should I use to remove my carbohydrate under native conditions?
  5. How do I inhibit O-Glycosidase?
  6. What is a good positive control for O-Glycosidase?
  7. 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?
  8. Do detergents inhibit O-Glycosidase?
  9. I tried using O-Glycosidase on my glycoprotein and didn't see removal of the carbohydrate. What could be the problem?

Quality, Safety & Legal

Quality Assurance Statement

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.

Certificate Of Analysis

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

Safety DataSheets

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

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@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.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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