Product Class: Other

O-Glycosidase
NEBU cloned at NEB recombinant 37 65 Heat

Product Introduction

O-Glycosidase, also known as Endo-α-N-Acetylgalactosaminidase, catalyzes the removal of Core 1 and Core 3 O-linked disaccharides from glycoproteins.

  • Can be used in conjunction with other exoglycosidases to remove more complex O-glycans
  • Recombinant enzyme with no detectable exoglycosidase or other endoglycosidase 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
P0733S 2000000.0 units 40000000 units/ml
P0733L 1.0E7 units 40000000 units/ml

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Product Information

Description


 
O-Glycosidase, also known as Endo-α-N-Acetylgalactosaminidase, catalyzes the removal of Core 1 and Core 3 O-linked disaccharides from glycoproteins.

Substrate Specificity:

Substrate Specificity:

Product Source

Cloned from Enterococcus faecalis and expressed in E. coli (1).
This product is related to the following categories:
Endoglycosidases Products,
Proteome Analysis Products,
This product can be used in the following applications:
Expression Systems,
Protein Digestion,
Glycan Sequencing,
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
  • P0733S     -20    
      O-Glycosidase P0733SVIAL -20 1 x 0.05 ml 4.0E7 units/ml
      GlycoBuffer 2 B3704SVIAL -20 1 x 1 ml 10 X
      Glycoprotein Denaturing Buffer B1704SVIAL -20 1 x 1 ml 10 X
      NP-40 B2704SVIAL -20 1 x 1 ml 10 %
  • P0733L     -20    
      O-Glycosidase P0733LVIAL -20 1 x 0.25 ml 4.0E7 units/ml
      GlycoBuffer 2 B3704SVIAL -20 1 x 1 ml 10 X
      Glycoprotein Denaturing Buffer B1704SVIAL -20 1 x 1 ml 10 X
      NP-40 B2704SVIAL -20 1 x 1 ml 10 %

Properties & Usage

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).

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)

Storage Buffer

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

Heat Inactivation

65°C for 10 minutes

Molecular Weight

Apparent: 147000 daltons

Unit Assay Conditions

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 (1).

Application Features

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

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.
  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.

References

  1. Morgan, W.T.J. and Elson, L.A. (1994). Biochem. J.. 28, 988-995.

Protocols, Manuals & Usage

Protocols

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

Application Notes

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  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. Do detergents inhibit O-Glycosidase?
  5. Can Neuraminidase be used together in a digest with PNGase F and O-Glycosidase?
  6. Can I double digest PNGase F and O-Glycosidase?
  7. What is the difference between PNGase F, Endo H and O-Glycosidase?
  8. 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?
  9. What are Glycosidases and their uses?
  10. Is it necessary to treat my glycoprotein concomitantly with Neuraminidase and O-Glycosidase?
  11. How do I inhibit O-Glycosidase?

Tech Tips

Don’t forget to include Neuraminidase (P0720) in your reaction! 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.

NEB’s O-Glycosidase is the only available O-Glycosidase that catalyzes the removal of Core 1 and Core 3 O-linked disaccharides from glycoproteins

You can use this enzyme under native or denaturing conditions

Under native conditions we recommend adding more enzyme and using longer incubation times

Under denaturing conditions the enzyme activity is increased two-fold. However, this observation may be substrate dependent.

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.

Specifications

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]

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 [email protected].

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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